Novel alkaline protease variants and detergents and cleaning agents containing said novel alkaline protease variants

ABSTRACT

Described herein are novel alkaline protease variants derived from subtilisin. These variants have, with respect to the amino acid sequence of  Bacillus lentus  subtilisin, variations at amino acid positions 199 and 211, and at least one modification that contributes to the stabilization of the molecule, the modification preferably being variations at amino acid positions 3 and/or 4. Preferably, the variant is  B. lentus  alkaline protease S3T/NV4I/V199I/L211G. Also described are detergents and cleaning agents comprising the novel alkaline protease variants. Methods of use employing the novel alkaline protease variants are also described.

The present invention relates to novel alkaline protease variants whichare derived from natural or modified subtilisin proteases. According tothe numbering of the subtilisin from Bacillus lentus, said variantshave, compared to the previously known subtilisins, the two amino acidpositions 199I and 211G and at least one modification, preferably, afterpoint mutation, the amino acids threonine in position 3 and/orisoleucine in position 4, which modification contributes to thestabilization of the molecule. Particular preference is given to thevariant B. lentus alkaline protease S3T/V4I/V199I/L211G. These variantsdistinguish themselves from other protease variants by an improvedcontribution to the cleaning performance of detergents and cleaningagents. Therefore, in addition to said enzymes, the present inventionrelates to their use in various technical processes and, in particular,to detergents and cleaning agents containing said novel alkalineprotease variants.

Proteases of the subtilisin type (subtilases, subtilopeptidases, EC3.4.21.62) are classed as belonging to the serine proteases, due to thecatalytically active amino acids. They are naturally produced andsecreted by microorganisms, in particular by Bacillus species. They actas unspecific endopeptidases, i.e. they hydrolyze any acid amide bondslocated inside peptides or proteins. Their pH optimum is usually withinthe distinctly alkaline range. A review of this family is provided, forexample, in the paper “Subtilases: Subtilisin-like Proteases” by R.Siezen, pages 75-95 in “Subtilisin enzymes”, edited by R. Bott and C.Betzel, New York, 1996. Subtilisins are suitable for a multiplicity ofpossible technical uses, in particular as active ingredients ofdetergents or cleaning agents.

Apart from enzymes such as, for example, amylases, lipases orcellulases, proteases have already been used for decades as activecomponents in detergents and cleaning agents. They have the ability tobreak down proteinaceous soilings on the material to be cleaned such as,for example, textiles or dishes. Owing to their relatively highsolubility, the hydrolysis products are washed away with the wash liquoror are attacked, dissolved, emulsified or suspended by the othercomponents of the detergents or cleaning agents. Thus, synergisticeffects between the enzymes and the other components of the detergentsand cleaning agents in question can arise.

Owing to their favorable enzymic properties such as stability or pHoptimum, subtilisins stand out among the detergent and cleaning agentproteases. The most important subtilisin proteases currently used indetergents, which are partly natural molecules, partly variants derivedfrom these wild-type enzymes by mutagenesis, are listed below.

Subtilisin BPN′ which originates from Bacillus amyloliquefaciens and B.subtilis, respectively, has been disclosed in the studies by Vasantha etal. (1984) in J. Bacteriol. Volume 159, pp. 811-819 and by J. A. Wellset al. (1983) in Nucleic Acids Research, Volume 11, pp. 7911-7925. Thepatent applications WO 95/07991 and WO 95/30010 present, for example,variants with reduced binding to the substrate at a simultaneouslyincreased rate of hydrolysis, which were obtained as a result of pointmutations in the loop regions of said enzyme. The patent application WO95/29979, for example, discloses detergents containing such BPN′variants.

Two of the amino acid positions considered in the present patentapplication, namely positions 3 and 4, are not located in loop regions;the residues 199 and 211 are homologous to positions 205 and 217,respectively, of BPN′, which are located in loop 6 of the molecule, asis described in the applications mentioned, for example. Said loop isinvolved in substrate binding. Position 205 in BPN′ contains anisoleucine (I) by nature. Application WO 95/07991 proposes numerouspossible amino acids with which the tyrosine (Y) located by nature inposition 217 can be replaced, but not in conjunction with stabilizingmutations of the molecule; if a 217G variant is claimed, then only inconnection with other, catalytically compensating point mutations inthis substrate-binding loop. The only variants actually disclosed (p.14) with replacements in positions 205 and/or 217 are those in whichboth residues have been replaced with space-filling, usually alsoaliphatic amino acids. The same applies to application WO 95/29979. Whenpossessing a mutation in loop 6, the subtilisins of application WO95/30010 have at least one further mutation in a different loop.Examples of 217G variants which are disclosed are Y217G/S188D or thosewith even more replacements in other loops than loop 6, whose homologousamino acids are unchanged in the variants of the invention.

Subtilisin BPN′ serves as reference enzyme of the subtilisins, inparticular with respect to numbering of positions. Thus, for example,the point mutations of application EP 130756 which refer to allsubtilisins are also indicated with BPN′ numbering. These also includeposition 217 which corresponds to position 211 in the enzyme of theinvention. Further relevant positions have not been described previouslyby this document.

The publications by E. L. Smith et al. from 1968 in J. Biol. Chem.,Volume 243, pp. 2184-2191 and by Jacobs et al. (1985), Nucl. Acids Res.,Volume 13, pp. 8913-8926 introduce the protease subtilisin Carlsberg. Itis naturally produced by Bacillus licheniformis and obtainable under thetrade name Alcalase® from Novozymes A/S, Bagsværd, Denmark. Variantsthereof which are obtainable by point mutations and have reduced bindingto the substrate with a simultaneously increased rate of hydrolysis aredisclosed, for example, by application WO 96/28566. As in the BPN′applications discussed above, these are variants in which single ormultiple exchanges in the loop regions of the molecule have been carriedout; a variant 204I/216G (Carlsberg numbering) has not been describedpreviously therein.

The PB92 protease is produced naturally by the alkaliphilic bacteriumBacillus nov. spec. 92 and obtainable under the trade name Maxacal® fromGist-Brocades, Delft, The Netherlands. Its original sequence isdescribed in patent application EP 283075. Variants of said enzyme whichhave been obtained by point mutation and which are suitable for use indetergents and cleaning agents are disclosed in applications WO 94/02618and EP 328229. From the first of these, only the variant L211G/N212D hasa replacement identical to that of the variant claimed herein; norelevant variant emerges from the second application.

The subtilisins 147 and 309 are sold by Novozymes under the trade namesEsperase® and Savinase®, respectively. They are derived from Bacillusclausii strains which are disclosed by application GB 1243784. Variantsof said enzymes, which have been developed by means of point mutagenesiswith respect to usage in detergents and cleaning agents are disclosed,for example, in applications WO 89/06279, WO 95/30011, WO 99/27082 andWO 00/37599.

Application WO 89/06279 aims at achieving higher oxidation stability, anincreased rate of proteolysis and enhanced washing performance of theprotease. It reveals (p. 14) that only replacements at particularpositions should alter the physical or chemical properties of subtilisin147 or 309 molecules; the positions 3, 4 and 211 are not included here.Application WO 95/30011 introduces variants of subtilisin 309 which havepoint mutations in the loop regions of the molecule and thus exhibitreduced adsorption to the substrate with a simultaneously increased rateof hydrolysis; they do not include any mutations in positions 3 or 4;the only point mutation actually corresponding to the variant of thepresent application is the L211G substitution which, however, does notcorrelate in any case with a V199I substitution. Application WO 99/27082develops variants of, by way of example, subtilisin 309, whose washingperformance is enhanced by enlarging the active loops by inserting twoor more amino acids. Thus, they are not substitutions like in thepresent application.

Further examples of proteases established for use in detergents andcleaning agents are:

-   -   protease 164-A1 from Chemgen Corp., Gaithersburg, Md., USA, and        Vista Chemical Company, Austin, Tex., USA (WO 93/07276),        obtainable from Bacillus spec.;    -   Bacillus sp. PD138 NCIMB 40338 alkaline protease from Novozymes        (WO 93/18140);    -   proteinase K-16 from Kao Corp., Tokyo, Japan (U.S. Pat. No.        5,344,770), derived from Bacillus sp. ferm. BP-3376;    -   subtilisin DY, described by Nedkov et al. 1985 in Biol. Chem        Hoppe-Seyler, Volume 366, pp. 421-430, which has been optimized,        in particular for usage in detergents and cleaning agents, by        application WO 96/28557, again via specific point mutations in        the active loops, but not including a V204I variant        (corresponding to position 199 in the enzyme of the invention)        either alone or in combination with other substitutions; and    -   thermitase produced by Thermoactinomyces vulgaris (Meloun et        al., FEBS Lett. 1983, pp. 195-200) and optimized, for example,        according to application WO 96/28558.

Among numerous possible variants of thermitase, the document mentionedlast also describes the variants with the L221G substitution(corresponding to position 211 in the enzyme of the invention). Sincethe enzyme by nature has isoleucine at position 209 (corresponding to199 in the enzyme of the invention), two of the amino acid residuesimportant for the invention at the corresponding positions(corresponding to 199I/L211G) have hereby been previously described, butwithout the additional feature of additionally stabilizing the moleculein the presence of said two amino acids at said positions. Inparticular, no stabilizations by threonine in position 3 and/orisoleucine at position 4 (according to B. lentus alkaline protease) havebeen described previously. However, thermitase has the amino acidresidues serine and arginine at positions 10 and 11 which are homologousto said two positions of B. lentus alkaline protease (compare alignmentin WO 91/00345).

Moreover, thermitase is a molecule whose sequence over-all deviatesconsiderably from those of the other subtilisins (Meloun et al., p.198). Thus the homology between the mature proteins thermitase and B.lentus alkaline protease is 45% identity (62% similar amino acids).Something similar applies to proteinase K (WO 96/28556) whose homologyto B. lentus alkaline protease is only 33% identity (46% similar aminoacids) at the mature protein level.

The applications EP 199404, EP 251446, WO 91/06637 and WO 95/10591, forexample, describe further proteases which are referred to by Procter &Gamble Comp., Cincinnati, Ohio, USA as “protease A”, “protease B”,“protease C” and “protease D”, respectively, and which may be used indetergents and cleaning agents (compare WO 00/47707, p. 73). Theproteases of application EP 199404 are various variants which are basedon patent EP 130756, but which have no variations at the positionsrelevant to the present application (compare EP 199404 A2, column 20).Example 10 of patent EP 251446 B1 (p.49) demonstrates that Y217Gvariants are less stable than the wild-type enzyme and, therefore, thissubstitution is not pursued any further. According to application WO91/06637, “proteases C” are distinguished by point mutations atpositions 123 and 274. According to WO 95/10591, all “protease D”variants carry mutations at position 76 which is unchanged in thepresent protease and identical to that of BPN′; the same applies to theapplication and, respectively, patents WO 95/10615, U.S. Pat. Nos.6,017,871 and 6,066,611.

Other known proteases are the enzymes obtainable under the trade namesDurazym®, Relase®, Everlase®, Nafizym, Natalase® and Kannase® fromNovozymes, under the trade names Purafect®, Purafect OxP® and Properase®from Genencor, under the trade name Protosol® from Advanced BiochemicalsLtd., Thane, India and under the trade name Wuxi® from Wuxi SnyderBioproducts Ltd., China.

In order to enhance the washing performance of subtilisins, numerousapplications pursued the strategy of inserting additional amino acidsinto the active loops, thus, for example, apart from the applicationsalready mentioned, also the applications published with the numbers WO00/37599, WO 00/37621 to WO 00/37627 and WO 00/71683 to WO 00/71691.

Another strategy is to alter the surface charges of the molecule. Thus,for example, the applications WO 91/00334, WO 91/00335, WO 91/00345, EP479870, EP 945502 and EP 563103, introduce numerous amino acidsubstitutions which can be used to increase or decrease the isoelectricpoint of said molecules. From this, application WO 00/24924 derives amethod for identifying appropriately suitable variants. The same applieswith respect to WO 96/34935 according to which it is also possible tovary the hydrophobicity of said molecules according to the sameprinciple.

Another strategy for improving the washing performance of subtilisins isto randomly introduce point mutations into known molecules and to testthe variants obtained for their contributions to the washingperformance. This strategy is pursued, for example, by patent U.S. Pat.No. 5,700,676 in which the only position described which is relevant tothe present invention is a substitution at position 217 (BPN′numbering), in each case in addition to a plurality of othersubstitutions. The same also applies to patents U.S. Pat. Nos.5,310,675, 5,801,038, 5,955,340 and applications WO 99/20723 and WO99/20727. The only mutation proposed in patent U.S. Pat. No. 4,760,025,which is relevant to the present invention, is one at position 217, thereason for which is that said mutation affects the active site. All ofthese documents do not suggest that the other substitutions of theinvention could play a part with respect to washing performance.

A modern direction in enzyme development is to combine, via statisticalmethods, elements from known proteins related to one another to novelenzymes having properties which have not been achieved previously.Methods of this kind are also summarized under the generic term directedevolution and include, for example, the following methods: The StEPmethod (Zhao et al. (1998), Nat. Biotechnol., Volume 16, p. 258-261),Random priming recombination (Shao et al., (1998), Nucleic Acids Res.,Volume 26, p. 681-683), DNA shuffling (Stemmer, W. P. C. (1994), Nature,Volume 370, p. 389-391) or RACHITT (Coco, W. M. et al. (2001), Nat.Biotechnol., Volume 19, p. 354-359).

Another, in particular complimenting, strategy is to increase thestability of the proteases concerned and thus to increase theirefficacy. For example, U.S. Pat. No. 5,230,891 has described astabilization of this kind for proteases used in cosmetics. Fordetergents and cleaning agents, on the other hand, stabilizations bypoint mutations are more familiar. Thus, according to U.S. Pat. Nos.6,087,315 and 6,110,884, proteases can be stabilized by replacingparticular tyrosine residues with other residues. Other possibilitiesare, for example:

-   -   replacing particular amino acid residues with proline, according        to EP 583339;    -   introducing more polar or charged groups on the molecule        surface, according to EP 995801;    -   altering the binding of metal ions, in particular calcium        binding sites, for example according to the teaching of        applications WO 88/08028 and WO 88/08033;        further possibilities of stabilizing subtilisins, in particular        those derived from that of Bacillus lentus, are reported in        patents U.S. Pat. Nos. 5,340,735, 5,500,364, 5,985,639 and        6136553.

The B. lentus alkaline proteases are highly alkaline proteases ofBacillus species. One of these strains has been deposited under numberDSM 5483 (WO 91/02792, and, respectively, EP 493398 and U.S. Pat. No.5,352,604). WO 92/21760, WO 95/23221 and WO 98/30669 disclose variantsof this enzyme to be obtained by point mutation and usable in detergentsand cleaning agents.

The wild-type enzyme is derived from a producer which had originallybeen obtained by screening for alkaliphilic Bacillus strains anddisplays itself a comparatively high stability to oxidation and theaction of detergents. The applications WO 91/02792 and, respectively, EP493398 and U.S. Pat. No. 5,352,604 describe its heterologous expressionin the host Bacillus licheniformis ATCC 53926. The claims of said USpatent refer to positions 208, 210, 212, 213 and 268, but not to anyvariant having substitutions in positions 61 and 211, as beingcharacteristic for B. lentus alkaline protease.

Application WO 92/21760 also discloses the amino acid sequence, underSEQ ID NO:52, and the nucleotide sequence, under SEQ ID NO:106, of theB. lentus alkaline protease wild-type enzyme. In addition, thisapplication discloses 51 different variants which differ from thewild-type in numerous positions, among them also S3T, V4I and V199I.

The applications WO 95/23221 and WO 98/30669 also reveal B. lentusalkaline protease variants suitable for usage in detergents and cleaningagents, which correspond to the enzyme of the invention in the threepositions S3T, V4I and V199I. In addition, they all have two or threefurther point mutations compared to the wild-type enzyme from the B.lentus DSM 5483. Some of them carry an additional mutation at position211, namely 211D (variants F49, F54 and F55); consequently, saidapplications claim the substitutions 211D and 211E.

As all of these studies which have been carried out over a long periodof time confirm, there is high demand for alternative proteases forusage in detergents and cleaning agents. The most recent publicationssuch as, for example, WO 00/71683 to WO 00/71691, prove that even thelong established family of subtilisin proteases is still in need ofoptimization with respect to their usability in detergents and cleaningagents. Said need of optimization is accompanied by numerous studies onvariation in the amino acid sequence of the enzymes concerned. However,the behavior of said enzymes in the context of a detergent or cleaningagent formulation cannot be readily inferred from the possiblycalculatable enzymic properties (compare U.S. Pat. Nos. 5,801,039,5,985,639 and 6,136,553). Other factors, such as stability to oxidizingagents, denaturation by surfactants, folding effects or desiredsynergies with other ingredients, play a part here.

It was the object of the present invention to find subtilisins whichshow improved performances in technical applications. In particular, itwas intended to find those subtilisins which improve the washing orcleaning performance of detergents and/or cleaning agents.

Part of the object had been not only to improve the proteases withrespect to their hydrolytic activity but also to maintain theirstability in appropriate detergent and cleaning agent formulations.

With respect to this problem, the present patent application pursued thestrategy of further improving the Bacillus lentus DSM 5483 subtilisin,in particular compared to the molecules disclosed in applications WO91/02792, WO 92/21760 and WO 95/23221, for usage in detergents andcleaning agents.

Surprisingly, it was found that the amino acids isoleucine and glycineat positions 199 and 211 result in an increased washing performancecontribution which is enhanced, presumably via a stabilizing effect, bythe amino acids threonine and isoleucine at positions 3 and 4,respectively.

According to the invention, this object is thus achieved by alkalineproteases of the subtilisin type, which are characterized in that,according to the numbering of Bacillus lentus DSM 5483 subtilisin, theyhave isoleucine at position 199 and glycine at position 211 and at leastone stabilization, preferably due to the amino acids threonine atposition 3 and/or isoleucine at position 4.

It is likewise achieved by subtilisin variants which are characterizedin that, according to the numbering of Bacillus lentus DSM 5483subtilisin, they have isoleucine at position 199 and glycine at position211 and, more preferably, additionally one or both of the amino acidsthreonine at position 3 and isoleucine at position 4.

It is likewise achieved by subtilisin variants which are characterizedin that, according to the numbering of Bacillus lentus DSM 5483subtilisin, they have threonine at position 3, isoleucine at position 4,isoleucine at position 199 and glycine at position 211.

It is particularly achieved by appropriate alkaline proteases of thesubtilisin type which are characterized in that they are naturallyproduced by a bacillus or can be derived from such a subtilisin, inparticular of Bacillus lentus.

Very particularly, it is achieved by alkaline proteases which arenaturally produced by Bacillus lentus DSM 5483 or can be derived fromsuch alkaline proteases, and among these in particular B. lentusalkaline protease S3T/V4I/V199I/L211G according to the amino acidsequence indicated in SEQ ID NO.4.

The B. lentus alkaline protease variant M131 with the characterizingsubstitutions S3T/V4I/A188P/V193M/V199I must be regarded as the variantfrom WO 92/21760, which has the highest degree of homology to the B.lentus alkaline protease variant of the invention, S3T/V4I/V199I/L211G.It corresponds in three positions to those of the variant of theinvention. The difference is the two substitutions A188P and V193M atwhose positions the variant of the invention is identical to the wildtype. As, for example, application WO 95/30011 demonstrates, amino acid193 of B. lentus subtilisins is located at the start of loop 6, whileamino acid 188 is to be assigned not to any loop but to the compactprotein region located in between. In this respect, both mutations arelocated in structurally different regions of the molecule. Surprisingly,it was found in. the present invention that reversing the two positions188 and 193 to the wild-type amino acids and an additional mutation inposition 211, i.e. in the posterior region of loop 6, results in anenzyme which is superior to the previously known enzymes, in particularthe previously known variants of B.lentus alkaline protease, withrespect to its washing and cleaning performance.

The particularly preferred enzyme of the invention differs from thevariants of applications WO 95/23221 and WO 98/30669 in that a pluralityof positions have reverted, i.e. are identical again to the wild type,and that position 211 contains the non-space-saving and uncharged aminoacid glycine instead of the leucine of the wild type or the aspartate ofsaid variants.

From an enzymological point of view, it is surprising that the effect ofimproved washing and cleaning performance is achieved by a substitutionin an amino acid which is presumably involved in substrate bindingand/or catalysis of the reaction; namely by replacing the space-saving,hydrophobic side chain of leucine with the side chain of a glycine,which is reduced to a proton. At the same time, the second position ofloop 6, which had been mutated compared to the wild type, namely 199,need not be reverted from isoleucine to valine of the wild type. Incomparison to the applications WO 95/23221 or WO 98/30669, the pointmutagenesis to give an acidic group, i.e. L211D or L211E, would havebeen more obvious than reversion to the wild-type sequence at the othermutated positions. The documents, cited at the outset, for variation ofthe active loops of the various subtilisins, in particular WO 95/30011,would, with variation of position 211, have suggested an additionalchange in another active loop or a more drastic change within the sameloop, such as, for example, V199S/L211D, P204E/L211G or G196S/L211G, inorder to compensate for the change catalytically, but by no meanssticking to the V199I substitution.

It is surprising, from the viewpoint of application, in particular indetergents and cleaning agents, that this results in performanceimprovement, in particular in an improvement of contribution of suchenzymes to the washing and cleaning performance on a large variety ofsoilings. The successful use of subtilisins of the invention inappropriate washing and cleaning agent formulations (compare Examples 2to 5) suggests that the stability of the variants concerned is also highenough in order to keep the enzymes active for a sufficiently longperiod and has thus contributed to improved performance.

The present invention relates to an alkaline protease of the subtilisintype, characterized in that, according to the numbering of Bacilluslentus DSM 5483 substilisin, it has isoleucine at position 199 andglycine at position 211 and at least one stabilization. Preferably, saidstabilization is additionally one of the amino acids threonine atposition 3 or isoleucine at position 4.

Further embodiments of this subject matter of the invention are alkalineproteases of the subtilisin type, characterized in that, according tothe numbering of Bacillus lentus DSM 5483 subtilisin, they havethreonine at position 3, isoleucine at position 4, isoleucine atposition 199 and glycine at position 211; that they are subtilisinsnaturally produced by a Bacillus, in particular by Bacillus lentus, orderived from such a Bacillus; that they are subtilisins naturallyproduced by or derived from Bacillus lentus DSM 5483, in particular B.lentus alkaline protease S3T/V4I/V199I/L211G according to the amino acidsequence indicated in SEQ ID NO.4.

Further embodiments of this subject matter of the invention are proteinsderived from corresponding alkaline proteases of the subtilisin type, inparticular by fragmentation or deletion mutagenesis, by insertionmutagenesis, by substitution mutagenesis or by fusion of at least onepart to at least one other protein; those additionally characterized inthat they are additionally derivatized; that they have a proteolyticactivity, preferably an increased proteolytic activity compared to thestarting molecule and, respectively, nonderivatized molecule, and veryparticularly enhanced performance; and/or that they are additionallystabilized.

The invention further relates to nucleic acids which code for theproteins referred to in the first subject matter of the invention, inparticular nucleic acids coding for subtilisin proteases, whosenucleotide sequence corresponds to the nucleotide sequence indicated inSEQ ID NO.3, in particular in the regions coding for 199 isoleucine and211 glycine and very particularly in the regions coding for 3 threonine,4 isoleucine, 199 isoleucine and 211 glycine.

The present invention further relates to vectors which contain a nucleicacid region as defined above and comprises, in particular, a nucleicacid region coding for any of the proteins or derivatives as defined inthe first subject matter of the invention. They are, in preferredembodiments, cloning vectors which comprise a nucleic acid region asdefined above and which comprise, in particular, a nucleic acid regioncoding for any of the proteins or derivatives as defined in the firstsubject matter of the invention; or they are expression vectors whichcomprise a nucleic acid region as defined above and which comprise, inparticular, a nucleic acid region coding for any of the proteins orderivatives as defined in the first subject matter of the invention andmaking possible the biosynthesis thereof.

The invention further relates to cells which comprise a vector accordingto the abovementioned subject matter of the invention; which preferablyexpress or can be induced to express any of the proteins or derivativesas defined in the first subject matter of the invention, in particularby using an expression vector as defined above; which are preferablycharacterized in that they are bacteria, in particular those whichsecrete the protein produced into the surrounding medium; which arepreferably characterized in that they are bacteria of the genusBacillus, in particular of the species Bacillus lentus, Bacilluslicheniformis, Bacillus amyloliquefaciens, Bacillus subtilis or Bacillusalcalophilus; or which are characterized in that they are eukaryoticcells, in particular those which modify posttranslationally the producedprotein.

The invention further relates to methods for preparing a proteolyticenzyme or derivative according to the first subject matter of theinvention by using a host cell as defined above and/or using a vector asdefined above and/or using a nucleic acid as defined above.

The invention further relates to agents which are characterized in thatthey comprise proteolytic enzymes according to the first subject matterof the invention, in particular detergents or cleaning agents, veryparticularly in an amount of from 2 μg to 20 mg per g of agent;preferably those which are characterized in that they additionallycomprise further enzymes, in particular other proteases, amylases,cellulases, hemicellulases and/or lipases.

The invention further relates to agents for the treatment of textile rawmaterials or for textile care, which are characterized in that theycontain either solely or in addition to other active ingredients, aproteolytic enzyme according to the first subject matter of theinvention, in particular for fibers or textiles containing naturalcomponents and, very particularly, for those containing wool or silk.

The invention further relates to methods for machine cleaning textilesor hard surfaces, which methods are characterized in that in at leastone of the method steps a proteolytic enzyme according to the firstsubject matter of the invention becomes active, preferably in an amountof from 40 μto 4 g, particularly preferably from 400 μto 400 mg, perapplication.

The invention further relates to methods for the treatment of textileraw materials or for textile care, which methods are characterized inthat in at least one of the method steps a proteolytic enzyme accordingto the first subject matter of the invention becomes active, inparticular for textile raw materials or textiles containing naturalcomponents, in particular for those containing wool or silk.

The invention further relates to uses of a proteolytic enzyme accordingto the first subject matter of the invention for cleaning textiles orhard surfaces, preferably in an amount of from 40 μto 4 g, particularlypreferably from 400 μto 400 mg, per application.

The invention further relates to uses of a proteolytic enzyme accordingto the first subject matter of the invention for activating ordeactivating ingredients of detergents or cleaning agents.

The invention further relates to uses of a proteolytic enzyme accordingto the first subject matter of the invention for biochemically analyzingor for synthesizing low molecular weight compounds or proteins.

The invention further relates to uses of a proteolytic enzyme accordingto the first subject matter of the invention for preparing, purifying orsynthesizing natural substances or biological valuable substances.

The invention further relates to uses of a proteolytic enzyme accordingto the first subject matter of the invention for the treatment ofnatural raw materials, in particular for the treatment of surfaces, veryparticularly in a method for the treatment of leather.

The invention further relates to uses of a proteolytic enzyme accordingto a the first subject matter of the invention for the obtainment ortreatment of raw materials or intermediates in the manufacture oftextiles, in particular for removing protective layers on fabrics.

The invention further relates to uses of a proteolytic enzyme accordingto the first subject matter of the invention for the treatment oftextile raw materials or for textile care, in particular for thetreatment of wool or silk or of wool- or silk-containing mixed textiles.

The invention further relates to uses of a proteolytic enzyme accordingto the first subject matter of the invention for the treatment ofphotographic films, in particular for removing gelatin-containing orsimilar protective layers.

The invention further relates to uses of a proteolytic enzyme accordingto the first subject matter of the invention for preparing food oranimal feed.

The invention further relates to cosmetics containing a proteolyticenzyme according to the first subject matter of the invention or tocosmetic methods including a proteolytic enzyme according to the firstsubject matter of the invention or to the use of a proteolytic enzymeaccording to the first subject matter of the invention for cosmeticpurposes, in particular within the framework of corresponding methods orin corresponding agents.

A protein means in accordance with the present application a polymerwhich is composed of the natural amino acids, has a substantially linearstructure and adopts usually a three-dimensional structure to exert itsfunction. Table 1 lists the 19 proteinogenic, naturally occurringL-amino acids, together with the 1- and 3-letter codes which are alsoused in the present application for abbreviation of said amino acids.TABLE 1 The proteinogenic amino acids 1-letter code 3-letter code Fullname A Ala alanine C Cys cysteine D Asp aspartic acid E Glu glutamicacid F Phe phenylalanine G Gly glycine H His histidine I Ile isoleucineK Lys lysine L Leu leucine M Met methionine N Asn asparagine P Proproline Q Gln glutamine R Arg arginine S Ser serine T Thr threonine VVal Valine W Trp tryptophane

The combination of any of these names/codes with a number indicates theamino acid residue which the particular protein carries at therespective position. Thus, for example, S3 indicates a serine residue atposition 3, starting with the numbering at the N terminus of the proteinin question. According to this nomenclature, a point mutation at thissite, for example to give the amino acid threonine, is abbreviated withS3T. In order to denote variants having a plurality of point mutations,these substitutions are separated from one another by forward slashes.Accordingly, the variant S3T/V4I is characterized in that the serinepreviously present at position 3 of said variant has been replaced witha threonine and the valine at position 4 has been replaced with anisoleucine.

Unless stated otherwise, the positions indicated in the presentinvention refer to the in each case mature forms of the proteinsconcerned, i.e. without the signal peptides (see below).

An enzyme in accordance with the present application means a proteinwhich exerts a particular biochemical function. Proteolytic enzymes orenzymes with proteolytic function, for example, mean generally thosewhich hydrolyze the acid amide bonds of proteins, in particular thosebonds located inside the proteins, and which may therefore also bereferred to as endopeptidases. Subtilisin proteases are thoseendopeptidases which are naturally produced by Gram-positive bacteriaand usually secreted or which are derived from the latter, for examplevia molecular biological methods, and can be homologized with thenatural subtilisin proteases via part regions such as structure-formingor function-carrying regions. They are described, for example, in thearticle “Subtilases: Subtilisin-like Proteases” by R. Siezen, pages75-95 in “Subtilisin enzymes”, edited by R. Bott and C. Betzel, NewYork, 1996.

Numerous proteins are formed as “preproteins”, i.e. together with asignal peptide. This then means the N-terminal part of the protein,whose function usually is to ensure the export of the produced proteinfrom the producing cell into the periplasm or into the surroundingmedium and/or the correct folding thereof. Subsequently, the signalpeptide is removed from the remaining protein under natural conditionsby a signal peptidase so that said protein exerts its actual catalyticactivity without the initially present N-terminal amino acids. Accordingto FIG. 1 in WO 91/02792, the preprotein of Bacillus lentus DSM 5483subtilisin contains 380 amino acids, the mature protein, however, only269; the numbering starts with the first amino acid of the matureprotein, i.e. in this case with the alanine which would have number 112according to the preprotein sequence. According to SEQ ID NO. 1 and 2,the signal peptide of B. licheniformis ATCC 68614 subtilisin is 111amino acids and the mature peptide 269 amino acids in length. Withoutthis division, the complete protein is 380 amino acids in length, as SEQID NO.2 reveals. According to SEQ ID NO.3 and 4, the same applies to theparticularly preferred embodiment.

Owing to their enzymic activity, preference is given for technicalapplications to the mature peptides, i.e. the enzymes processed aftertheir preparation, over the preproteins.

Pro-proteins are inactive precursors of proteins. Their precursors withsignal sequence are referred to as prepro-proteins.

Nucleic acids mean in accordance with the present application themolecules which are naturally composed of nucleotides, serve asinformation carriers and code for the linear amino acid sequence inproteins or enzymes. They may be present as single strand, as a singlestrand complementary to said single strand or as double strand. Formolecular-biological work, preference is given to the nucleic acid DNAas the naturally more durable information carrier. In contrast, an RNAis produced to implement the invention in a natural environment such as,for example, in an expressing cell, and RNA molecules important to theinvention are therefore likewise embodiments of the present invention.

In accordance with the present application, the information unit of anucleic acid, which corresponds to a protein, is also referred to asgene. In the case of DNA, the sequences of both complementary strands inin each case all three possible reading frames must be taken intoaccount. The fact that different codon triplets can code for the sameamino acids so that a particular amino acid sequence can be derived froma plurality of different nucleotide sequences which possibly have onlylow identity must also be taken into account (degeneracy of the geneticcode). Moreover, various organisms differ in the use of these codons.For these reasons, both amino acid sequences and nucleotide sequencesmust be incorporated into the scope of protection, and nucleotidesequences indicated are in each case regarded only as coding by way ofexample for a particular amino acid sequence.

It is possible for a skilled worker, via nowadays generally knownmethods such as, for example, chemical synthesis or polymerase chainreaction (PCR) in combination with molecular-biological and/orprotein-chemical standard methods, to prepare complete genes on thebasis of known DNA sequences and/or amino acid sequences. An idealstarting point for this are DNA preparations of deposited and/orcommercially available microorganisms. Such methods are known, forexample, from the “Lexikon der Biochemie [Encyclopedia ofBiochemistry]”, Spektrum Akademischer Verlag, Berlin, 1999, Volume 1,pp. 267-271 and Volume 2, pp. 227-229.

Changes of the nucleotide sequence, as may be produced, for example, bymolecular-biological methods known per se, are referred to as mutations.Depending on the type of change, deletion, insertion or substitutionmutations, for example, or those in which various genes or parts ofgenes are fused to one another (shuffling) are known; these are genemutations. The corresponding organisms are referred to as mutants. Theproteins derived from mutated nucleic acids are referred to as variants.Thus, for example, deletion, insertion, substitution mutations orfusions result in deletion-, insertion-, substitution-mutated or fusiongenes and, at the protein level, in corresponding deletion, insertion orsubstitution variants, or fusion proteins.

Vectors mean in accordance with the present invention elements whichconsist of nucleic acids and which contain a gene of interest ascharacteristic nucleic acid region. They are capable of establishingsaid gene as a stable genetic element replicating independently of theremaining genome in a species or a cell line over several generations orcell divisions. Vectors are, in particular when used in bacteria,special plasmids, i.e. circular genetic elements. Genetic engineeringdistinguishes between, on the one hand, those vectors which are used forstorage and thus, to a certain extent, also for genetic engineeringwork, the “cloning vectors”, and, on the other hand, those which performthe function of establishing the gene of interest in the host cell, i.e.enabling expression of the protein in question. These vectors arereferred to as expression vectors.

Homologization, i.e. comparison with known enzymes, as carried out viaan alignment, for example, makes it possible to deduce the enzymicactivity of an enzyme studied from the amino acid or nucleotidesequence. Said activity may be modified qualitatively or quantitativelyby other regions of the protein which are not involved in the actualreaction. This could concern, for example, enzyme stability, activity,reaction conditions or substrate specificity.

The term proteolytic enzyme or protease therefore means, in addition tothe functions of the few amino acid residues of the catalytically activesite, any functions as resulting from the action of the entire remainingprotein or one or more parts of the remaining protein on the actuallycatalytically active regions. In accordance with the invention, suchmodifying functions or part activities alone are also regarded asproteolytic activity, as long as they support a proteolytic reaction.Such auxiliary functions or part activities include, for example,binding of a substrate, an intermediate or an end product, theactivation or inhibition or mediation of a regulating influence on thehydrolytic activity. Another possible example is the formation of astructural element located far away from the active site. The secondprecondition for the fact that it is a protein of the invention,however, is that the chemical behavior of the actually active residuesalone or, in addition, the action of the modifying parts results in ahydrolysis of peptide bonds. It is furthermore possible that one or moreparts of, for example, the protein of the invention also modifyqualitatively or quantitatively the activities of other proteases. Thisinfluencing of other factors is regarded as proteolytic activity.Proteolytically active enzymes are also those whose activity at a givenpoint in time is blocked, for example by an inhibitor. Their principalsuitability for the corresponding proteolytic reaction is crucial.

Fragments mean any proteins or peptides which are smaller than naturalproteins or those which correspond to completely translated genes, andmay also be obtained synthetically, for example. Owing to their aminoacid sequences, they may be related to the corresponding completeproteins. They may adopt, for example, identical structures or exertproteolytic activities or partial activities such as complexing of asubstrate, for example. Fragments and deletion variants of startingproteins are in principle very similar; while fragments represent ratherrelatively small pieces, the deletion mutants rather lack only shortregions and thus only individual partial functions.

Chimeric or hybrid proteins mean in accordance with the presentapplication those proteins which are composed of elements whichnaturally originate from different polypeptide chains from the sameorganism or from different organisms. This procedure is also calledshuffling or fusion nmtagenesis. The purpose of such a fusion may be,for example, to cause or to modify an enzymic function with the aid ofthe fused-to protein part of the invention. In accordance with thepresent invention, it is unimportant as to whether such a chimericprotein consists of a single polypeptide chain or of a plurality ofsubunits between which different functions may be distributed. Toimplement the latter alternative, it is possible, for example, to breakdown a single chimeric polypeptide chain into a plurality of polypeptidechains by a specific proteolytic cleavage, either posttranslationally oronly after a purification step.

Proteins obtained by insertion mutation mean those variants which havebeen obtained via methods known per se by inserting a nucleic acidfragment or protein fragment into the starting sequences. They should beclassified as chimeric proteins, due to their similarity in principle.They differ from the latter merely in the size ratio of the unalteredprotein part to the size of the entire protein. In suchinsertion-mutated proteins the proportion of foreign protein is lowerthan in chimeric proteins.

Inversion mutagenesis, i.e. a partial sequence conversion, may beregarded as a special form of both deletion and insertion. The sameapplies to a regrouping of various molecule parts, which deviates fromthe original amino acid sequence. Said regrouping can be regarded asdeletion variant, as insertion variant and as shuffling variant of theoriginal protein.

Derivatives mean in accordance with the present application thoseproteins whose pure amino acid chain has been chemically modified. Thosederivatizations may be carried out, for example, biologically inconnection with protein biosynthesis by the host organism.Molecular-biological methods may be employed here. However, saidderivatizations may also be carried out chemically, for example bychemical conversion of an amino acid side chain or by covalent bindingof another compound to the protein. Such a compound may also be, forexample, other proteins which are bound, for example, via bifunctionalchemical compounds to proteins of the invention. Such modifications mayinfluence, for example, substrate specificity or the strength of bindingto the substrate or cause transient blocking of the enzymic activity ifthe coupled-to substance is an inhibitor. This may be useful for theperiod of storage, for example. Likewise, derivatization means covalentbinding to a macromolecular support.

In accordance with the present invention, all enzymes, proteins,fragments and derivatives, unless they need to be explicitly referred toas such, are included under the generic term proteins.

The performance of an enzyme means its efficacy in the technical areaconsidered in each case. Said performance is based on the actual enzymicactivity but, in addition, depends on further factors relevant for theparticular process. These include, for example, stability, substratebinding, interaction with the material carrying said substrate orinteractions with other ingredients, in particular synergies.

The washing or cleaning performance of an agent means in accordance withthe present application the effect exerted by the agent studied on thesoiled articles, for example textiles or objects with hard surfaces.Individual components of such agents, for example individual enzymes,are evaluated with respect to their contribution to the washing orcleaning performance of the entire agent, for it is not readily possibleto deduce the contribution of an enzyme to the washing performance of anagent from the enzymic properties of said enzyme. Examples of otherfactors which play a part here are stability, substrate binding, bindingto the material to be cleaned and interactions with other ingredients ofsaid agents, in particular synergies in removing the soilings.

According to the Budapest Treaty on the international recognition of thedeposit of microorganisms from Apr. 28, 1977, the followingmicroorganism has been deposited in connection with application WO91/02792, on Aug. 10, 1989, with the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH in Brunswick, Germany: Bacilluslentus DSM 5483. There it has the registration number DSM 5483(PA5-A0155, Strain 2-1). The essential information on the features ofthis biological material is summarized in WO 91/02792, Table 1 (pages 5to 7). The DNA sequence and amino acid sequence of the alkaline proteasefrom said organism, which is particularly relevant to the presentapplication, can be found under SEQ ID NO:106 and SEQ ID NO:52,respectively.

Particularly important to the invention are positions 3, 4, 199 and 211of the mature proteins according to the Bacillus lentus DSM 5483subtilisin numbering (WO 92/21760). These can be homologized accordingto Table 2 with those of the most important subtilisins; saidhomologization can be transferred to all other subtilisins. Thus, forexample, the article “Subtilases: Subtilisin-like Proteases” by R.Siezen, pages 75-95 in “Subtilisin enzymes”, edited by R. Bott and C.Betzel, New York, 1996 shows an alignment of more than 20 subtilisins inrelation to the known sequence of subtilisin BPN′. TABLE 2Homologization of the four positions particularly important to theinvention Numbering Reference according to the Pos. Pos. enzymessequences in Pos. 3 Pos. 4 199 211 B. lentus WO 92/21760 S 3 V 4 V 199 L211 alkaline protease BPN′ Wells et al. (see S 3 V 4 I 205 Y 217 above)Subtilisin Smith et al. (see T 3 V 4 V 204 L 216 Carlsberg above) PB92EP 283075 S 3 V 4 V 199 L 211 Subtilisin WO 89/06279 S 3 V 4 V 199 L 211309 Thermitase WO 91/00345 S 10 R 11 I 209 L 221 Proteinase K WO91/00345 T 4 A 6 I 208 I 220

FIG. 1 of the present patent application also depicts an alignment ofthe amino acid sequences of a B. lentus alkaline protease variant of theinvention with these most important subtilisins described at the outset,namely Subtilisin 309 (Savinase®), Subtilisin PB92, Subtilisin Carlsbergand Subtilisin BPN′.

Owing to the high structural homologies between the various knownsubtilisins and to the same reaction mechanism of hydrolyzing endogenousacid amide bonds, which is exerted by them, it can be expected that saidpoint mutations act in each case comparably in the context of themolecule in question. In particular, it can be expected, owing to theteaching of the present patent application, that such subtilisins whichhave already been developed in the prior art with regard to their usagein detergents and cleaning agents are improved further with respect totheir contributions to the washing and cleaning performances by adoptingthese point mutations.

Adopting the amino acids isoleucine 199 and glycine 211 at thehomologous positions, in particular, should contribute to improving thecontribution of enzymes known from the prior art to the washing andcleaning performance in appropriate agents. Owing to the experienceswith B.lentus alkaline protease, however, these substitutions willprofit from at least one additional stabilization of the moleculesconcerned.

The stability of inventive proteases having the amino acid positions199I and 211G may be increased, for example, by coupling to polymers.Such a method is described in U.S. Pat. No. 5,230,891, for example. Itrequires linking the proteins, prior to their use in appropriate agents,via a chemical coupling step to such polymers.

Preference is given to stabilizations possible via point mutagenesis ofthe molecule itself, since they do not require any further working stepsfollowing obtainment of the protein. Some point mutations suitable forthis are known per se from the prior art. Thus, according to U.S. Pat.Nos. 6,087,315 and 6,110,884, proteases may be stabilized by replacingparticular tyrosine residues with other residues. Applied to Bacilluslentus-derived proteins of the invention, this would mean substitutionsof the tyrosine residues at positions 89, 161, 165, 208 and 257,according to SEQ ID NO.2; the other two positions indicated there arealready occupied by tyrosine anyway in B. lentus alkaline protease.

Other Possibilities Are, for Example:

-   -   replacing particular amino acid residues with proline, according        to EP 583339; this would mean for enzymes derived from B.lentus        the substitutions S55P, A96P, A166P, A188P and/or S253P);    -   introducing more polar or charged groups on the surface of the        molecule, according to EP 995801;    -   altering the binding of metal ions, in particular the calcium        binding sites, for example according to the teaching of        applications WO 88/08028 and WO 88/08033. According to the first        of these documents, one or more of the amino acid residues        involved in calcium binding should be replaced with negatively        charged amino acids. According to the teaching of the second        document, point mutations should be introduced simultaneously in        at least one of the sequences of the two residues        arginine/glycine; this relates, for example in Bacillus lentus        subtilisins, to the NG sequences in positions 60/61, 115/116 and        212/213.    -   According to U.S. Pat. No. 5,453,372, proteins may be protected        by particular mutations on the surface against the effect of        denaturing agents such as surfactants; the positions indicated        there correspond to positions 134, 155, 158, 164, 188 and/or 189        in B. lentus alkaline protease. Further comparable possibilities        are indicated in U.S. Pat. Nos. 5,340,735, 5,500,364, 5,985,639        and 6,136,553.

In preferred embodiments, stabilization occurs due to the amino acidresidues of threonine at position 3 and/or of isoleucine at position 4,according to the Bacillus lentus DSM 5483 subtilisin numbering.

The variants studied in the examples of the present invention suggestthat their increased stability is the decisive factor for imparting tothem an improved washing performance, acting together with amino acids199I and 211G.

Independently of this theory, all alkaline proteases of the subtilisintype which are characterized in that, according to the numbering ofBacillus lentus DSM 5483 subtilisin, they have isoleucine at position199 and glycine at position 211 and additionally have either of the twoamino acids threonine at position 3 and isoleucin at position 4 aresolutions to the object of the invention.

In addition, preference is given to those variants which have, inaddition to 199 isoleucine and 211 glycine, both threonine at position 3and isoleucine at position 4.

Preference is given to corresponding variants of those alkalineproteases naturally produced by or derived from a Bacillus, sinceBacillus proteases have from the outset properties advantageous forvarious possible technical uses, including a certain stability to atemperature, oxidizing or denaturing agents. Moreover, most experiencehas been obtained with microbial proteases, with respect to theirbiotechnological production regarding, for example, construction ofsuitable cloning vectors, selection of host cells and fermentationconditions or evaluation of risks such as, for example, allergenicity.

Very particularly established in the prior art are the subtilisins ofBacillus lentus and the subtilisins derived from its naturally producedproteases, for example for use in detergents and cleaning agents. Theyinclude the proteases mentioned at the outset, subtilisin 147,subtilisin 309 and B.lentus alkaline protease. The wealth of experienceacquired for preparation and use of said proteases benefits furtherdevelopments of said enzymes according to the invention, including, forexample, their compatibility with other chemical compounds such as, forexample, the ingredients of detergents or cleaning agents.

A particularly preferred embodiment relates to the proteases of theinvention, which can be derived from those produced by Bacillus lentusDSM 5483. Included here are, for example, those of the variantsdescribed in applications WO 92/21760 and WO 95/23221 and WO 98/30669.Developments of these enzymes, which have the substitutions of theinvention at positions 199, 211, 3 and/or 4, characterize particularlypreferred embodiments of the present invention.

The B.lentus alkaline protease S3T/V4I/V199I/L211G studied in thepresent application is a development of B. lentus DSM 5483 subtilisinwhose amino acid sequence is disclosed under SEQ ID NO:52 and whosenucleotide sequence is disclosed under SEQ ID NO:106 in the sequencelisting of application WO 92/21760.

It was found that the contribution of this novel variant to an agentcorresponding to the washing and cleaning performance was higher thanthat of the comparable enzymes B.lentus alkaline protease F49 andSavinase® established in the prior art for these purposes (Examples2-5). The sequence listing lists the amino acid sequence of this variantunder number SEQ ID NO.2. The gene coding for this amino acid sequenceis listed in the sequence listing under number SEQ ID NO. 1. Owing tothe degeneracy of the genetic codes, numerous other nucleic acids arealso conceivable which likewise code for said variant and are equallypreferred alternatives within this subject matter of the invention.

A bacteria, in particular Bacillus, strain which produces the B.lentusalkaline protease S3T/V4I/V199I/L211G variant with the DNA sequences andamino acid sequences indicated in the sequence listing may be prepared,for example, following the method illustrated in Example 1 of thepresent application.

Further proteins can be derived via molecular-biological methodsestablished in the prior art from the alkaline proteases mentionedpreviously. Such methods are also discussed in detail in the textbookFritsch, Sambrook and Maniatis “Molecular cloning: a laboratory manual”,Cold Spring Harbour Laboratory Press, New York, 1989, for example.

Said further proteins include, for example, variants to which additionalproperties have been imparted via substitution mutagenesis or viafurther point mutations and which are, due to said additional propertiespredestined with respect to specific possible uses, for example due tochanges in surface charges, as disclosed in WO 00/36069, or due toalterations in the loops involved in catalysis or substrate binding, asdisclosed in WO 99/27082, for example. It is also possible to subjectlarger partial regions of said variants to mutagenesis. Thus it may bethe aim of fragment generation or deletion mutagenesis, for example, toselect specific partial functions of the protease or, on the other hand,to exclude them, for example substrate binding and the interactions withother compounds, exerted via particular regions of the molecule.

Insertion, substitution or fusion may provide proteases of the inventionwith additional functions. This includes possibly, for example, couplingto particular domains, such as binding to cellulose-binding domains, asdescribed in the publications WO 99/57154 to WO 99/57159, for example.The amino acid linkers denoted here may be constructed by forming anintegrated fusion protein of protease, linker region and binding domain.Such a binding domain could also come from the same or a differentprotease, for example in order to enhance binding of the protein of theinvention to a protease substrate. This increases the local proteaseconcentration, which increase may be advantageous in individualapplications, for example in the treatment of raw materials.

Said proteases may also be derivatized, in particular for optimizingthem for their particular target application. This includes chemicalmodifications, as described, for example, in application DE 40 13 142.They may also be modified, for example, by coupling of low or highmolecular weight chemical compounds, as are carried out by nature inconnection with protein biosynthesis by various organisms, such as, forexample, binding of a fatty acid radical close to the N terminus orglycosylations in synthesis by eukaryotic host cells. Proteolyticenzymes or fragments which are additionally derivatized are thusembodiments of the present invention.

In connection with the use of proteins of the invention in detergents orcleaning agents, coupling to other detersive substances or enzymes, forexample, is particularly useful. The patent applications WO 00/18865 andWO 00/57155, for example, describe comparable coupling approaches forcellulose-binding domains. Analogously, couplings to macromolecularcompounds such as, for example, polyethylene glycol may also be carriedout in order to modify the molecule with respect to further propertiessuch as stability or skin compatibility. U.S. Pat. No. 5230891, forexample, describes a modification of this kind for rendering theproteases in question more suitable for the use in cosmetics.

Derivatives of proteins of the invention can, in the broadest sense,also mean preparations of these enzymes. Depending on its obtainment,working-up or preparation, a protein may be associated with variousother substances, for example from the culture of the producingmicroorganisms, since culture supernatants of protease-producingmicroorganisms already exhibit a proteolytic activity, indicating thateven crude extracts may be used appropriately, for example forinactivating other proteinogenic activities.

A protein may also have been specifically admixed with particular othersubstances, for example to increase its storage stability. Therefore,any preparations of the actual protein of the invention are also inaccordance with the invention. This is also independent of whether ornot it actually produces said enzymic activity in a particularpreparation, since it may be desired that it has only low activity, ifany, during storage and produces its proteolytic function only whenused. This may depend, for example, on the folding state of the proteinor may result from the reversible binding of one or more accompanyingsubstances of the preparation to a protein of the invention. The jointpreparation of proteases with protease inhibitors, in particular, isknown from the prior art (WO 00/01826). Also included here are fusionproteins in which the inhibitors are bound via linkers, in particularamino acid linkers, to the particular proteases (WO 00/01831).

Said developments, derivatizations and preparations of proteins of theinvention are particularly desired if said proteins continue to beproteolytically active, since this is the precondition for theirpossible uses of the invention. Preferably, the proteases obtained byany kind of mutagenesis and/or derivatization have, compared to thestarting molecule and the non-derivatized molecule, respectively,increased proteolytic activity and very particularly improvedperformances with respect to their in each case intended technical fieldof use, including, in particular, improvement of their washing and/orcleaning performance for use in detergents or cleaning agents.

This is possible, for example, by combining the point mutations of theinvention with further point mutations which relate to the catalyticreaction, for example at the active site. Thus it would be possible,following the teaching of application WO 95/30011, for example, tomutate proteases of the invention which are those derived from Bacilluslentus subtilisin, in the loop regions or to introduce additional aminoacids. Such studies are described in the applications published undernumbers WO 00/37599, WO 00/37621 to WO 00/37627 and WO 00/71683 to WO00/71691.

The deletion of a region of the enzyme, which interacts with otheractive compounds in the reaction medium and thus impairs the overallreaction, for example via folding effects, could be such a desireddevelopment. Analogously, fusion to other active enzymes, for example toother proteases, is conceivable in order to achieve an increased rate ofhydrolysis.

The reversible blocking of a proteolytic activity during storage, due tobinding of an inhibitor, for example, can stop autoproteolysis and thuseffect a high rate of proteolysis in the reaction medium at the time ofdilution. Coupling to special binding domains, for example, may increasein the purification process the concentration of the protease close tothe substrate relative to that in the liquor and thus increase thecontribution of said enzyme to the performance of the agent.

Numerous possibilities of increasing the stability of enzymes, inparticular of those used in detergents and cleaning agents, are knownfrom the prior art (see above). Particularly relevant to the inventionamong these are, for practicability reasons, those methods which arebased on point mutagenesis. All of the possibilities already illustratedabove can also be applied in combination to variants of the invention,since, according to WO 89/09819, it can be assumed that multiplestabilizing mutations have an additive effect. Thus, variants of theinvention which have already been stabilized by either of or both of thetwo amino acids 3T and 4I, can be additionally stabilized by coupling toa polymer. They may, however, also have a stabilizing mutation at adifferent site of the molecule, for example due to substitution of oneor more of the tyrosine residues defined above, introduction ofparticular proline residues, alteration of surface charges or alterationof calcium-binding sites.

Nucleic acids are the starting point for virtually all commonmolecular-biological studies and developments of proteins and productionthereof, including, in particular, sequencing of the genes andderivation of the corresponding amino acid sequence, any type ofmutagenesis and expression of the proteins. As already mentioned above,such methods are described, for example, in the manual by Fritsch,Sambrook and Maniatis “Molecular cloning: a laboratory manual”, ColdSpring Harbour Laboratory Press, New York, 1989. The second subjectmatter of the invention are therefore nucleic acids coding for theproteins of the first subject matter of the invention or for derivativesthereof.

At the DNA level, the enzymes important to the invention may beoptimized for various applications via any methods generally listedunder the term “protein engineering”. This makes it possible, inparticular, to achieve the following properties which occur at theprotein level: improvement of the resistance of the derived protein tooxidation, of the stability to denaturing agents or proteases, to hightemperatures, to acidic or strongly alkaline conditions, alteration ofthe sensitivity to calcium or other cofactors, reduction inimmunogenicity or allergenic action.

Examples of mutated genes of the invention include those responsible forindividual, specific base substitutions or randomized point mutations,for deletions of individual bases or of partial sequences, fusions toother genes or gene fragments or inversions. Mutations or modificationsof this kind can predestine the enzyme derived from the respectivenucleic acids for specific applications. Such a mutagenesis may becarried out target-specifically or via random methods, for example usinga subsequent recognition and/or selection method (screening andselection) on the cloned genes, targeting the activity.

In particular for those nucleic acids coding for protein fragments, allthree reading frames, both in sense and in antisense orientation, mustbe taken into account, since such oligonucleotides can be used via thepolymerase chain reaction (PCR) as starting points for the synthesis ofrelated nucleic related acids. Such oligonucleotides are explicitlyincluded within the scope of protection of the present invention, inparticular when covering any of the regions corresponding to the fouramino acid positions 3, 4, 199 and/or 211. This applies also to thosewhich have variable sequences in exactly these positions so that, withina population of many primers, there may also be at least one that codesfor a partial sequence corresponding to SEQ ID NO.3 for such a position.The same applies to antisense oligonucleotides which may be used forregulating expression, for example.

The development of the proteases of the invention may be orientated inparticular on the ideas presented in the publication “Proteinengineering” by P. N. Bryan (2000) in Biochim. Biophys. Acta, Volume1543, pp. 203-222.

Preference is given to nucleic acids coding for subtilisin proteases,whose nucleotide sequence corresponds to the nucleotide sequenceindicated in SEQ ID NO.3. This applies particularly for the regionscoding for 199 isoleucine and 211 glycine and very particularly forthose coding for 3 threonine, 4 isoleucine, 199 isoleucine and 211glycine.

This preferentially applies to those which can be derived from asequence for a Bacillus lentus protease, and particularly, if they canbe derived from a sequence for a Bacillus lentus DSM 5483 protease. In avery particularly preferred case, the nucleic acid codes for theB.lentus alkaline protease S3T/V4I/V199I/L211G of the invention and/orcorresponds to the nucleotide sequence indicated in SEQ ID NO.3.

The scope of protection also includes, for example, those nucleic acidscoding for proteolytically active insertion or fusion mutants. Thus theregion responsible for this activity may be fused, for example, tocellulose-binding domains or may carry point mutations in catalyticallyinactive regions in order to enable the derived protein to be coupled toa polymer or to reduce the allergenicity thereof.

In order to handle the nucleic acids relevant to the invention, they areconveniently ligated into vectors. This includes, for example, vectorsderived from bacterial plasmids, from viruses or bacterial phages, orlargely synthetic vectors. They are suitable starting points formolecular-biological and biochemical studies of the gene in question, ofits expression or of the corresponding protein. Thus vectors containingthe nucleic acid molecules as defined above, in particular those codingfor the proteolytic enzymes as defined above are a subject matter of thepresent invention.

Cloning vectors are preferred embodiments of said subject matter of theinvention and are, in addition to storage, biological amplification orselection of the gene of interest, suitable for molecular-biologicalcharacterization of said gene. At the same time, they are transportableand storable forms of the claimed nucleic acids and are also startingpoints for molecular-biological techniques not linked to cells such as,for example, PCR or in-vitro mutagenesis methods. Preference is given tothose cloning vectors containing nucleic acid regions coding for theproteolytic enzymes as defined above.

Expression vectors of the invention are the basis for implementing thenucleic acids of the second subject matter of the invention inbiological production systems and thereby producing the proteins of thefirst subject matter of the invention. Preferred embodiments of saidsubject matter of the invention are expression vectors which carry allthe genetic elements necessary for expression, for example the naturalpromoter originally located upstream of said gene or a promoter fromanother organism. Said elements may be arranged, for example, in theform of an “expression cassette”. Preference is given to thoseexpression vectors comprising nucleic acid regions coding for theproteolytic enzymes as defined above.

Another possibility of implementing the present invention is cellscontaining a vector according to the third subject matter of theinvention. They are thus the microbiological dimension of the presentinvention by making possible, for example, amplification of theappropriate genes but also mutagenesis or transcription and translationthereof and, ultimately, biotechnological production.

Host cells which express or can be induced to express any of theproteins of the invention, thereby enabling biotechnological productionthereof, are an embodiment of said subject matter of the invention. Forthis purpose, they must have received, i.e. must have been transformedwith, the appropriate gene, conveniently via a vector. Said vector maybe present in the host cell extrachromosomally as separate geneticelement or may have been integrated into a chromosome and is preferablyany of the expression vectors as defined above.

Suitable host cells are in principle all organisms, i.e. prokaryotes,eukaryotes or Cyanophyta. Preference is given to those host cells whichare easily manageable genetically, with respect to, for example,transformation with the expression vector and to its stableestablishment, for example unicellular fungi or bacteria. Moreover,preferred host cells are distinguished by good microbiological andbiotechnological manageability. This relates, for example, to easyculturability, high growth rates, low requirements on fermentation mediaand good rates of production and secretion of foreign proteins.Frequently, it is necessary to determine experimentally the expressionsystems optimal for the individual case from the abundance of varioussystems available according to the prior art. In this way, any proteinof the invention can be obtained from a multiplicity of host organisms.

Preferred embodiments are those host cells whose activity can beregulated, owing to appropriate genetic elements, for example bycontrolled addition of chemical compounds, by changing the culturingconditions or as a function of the particular cell density. Thiscontrollable expression makes possible a very economical production ofthe proteins of interest.

In a preferred embodiment, the host cells are bacteria, in particularthose which secrete the protein produced into the surrounding medium,since bacteria distinguish themselves by short generation times and lowdemands on the culturing conditions. This makes it possible to establishcost-effective methods. Gram-negative bacteria such as, for example, E.coli, secrete a multiplicity of proteins into the periplasmic space.This may be advantageous for special applications. In contrast,Gram-positive bacteria such as, for example, bacilli release secretedproteins immediately into the nutrient medium surrounding the cells,from which the expressed proteins of the invention can be purifieddirectly, according to another preferred embodiment. Application WO01/81597 even discloses a method according to which it is achieved thatGram-negative bacteria also export the expressed proteins.

One embodiment of the present invention utilizes Bacillus lentus DSM5483 itself in order to (homologously) express proteins of theinvention. On the other hand, however, preference is given toheterologous expression. Bacteria preferred for heterologous expressioninclude those of the genus Bacillus, in particular those of the speciesBacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis orother species or strains of Bacillus alcalophilus, since these producecomparable subtilisins themselves so that it is possible to obtain viathis method a mixture of proteins of the invention with the subtilisinsendogenously produced by the host strains. Application WO 91/02792 (EP493398 B1) describes, for example, coexpression of this kind of B.lentusalkaline protease in Bacillus licheniformis ATCC 53926; numerouspossible expression vectors can also be found there. It can be expectedthat the newly found variants of the invention, in particular those ofBacillus lentus and very particularly B.lentus alkaline proteaseS3T/V4I/V199I/L211G can be prepared with the aid of said vectors and/orin said host system.

In another preferred embodiment of this subject matter of the invention,the host cells are eukaryotes, in particular those which modify theproduced protein posttranslationally. Examples of suitable eukaryotesare fungi such as actinomycetes or yeasts such as Saccharomyces orKluyveromyces. The modifications which such systems carry out, inparticular in connection with protein synthesis, include binding of lowmolecular weight compounds such as membrane anchors or oligosaccharides,for example. Oligosaccharide modifications of this kind may be desirablefor reducing the allergenicity of the prepared proteins, for example.

Methods for preparing a proteolytic enzyme or derivative of theinvention are a separate subject matter of the invention. Thus it ispossible, for example on the basis of the above-defined DNA sequencesand amino acid sequences, as can be derived, for example, also from thesequence listing, to synthesize corresponding oligopeptides andoligonucleotides up to the complete genes and proteins according tomolecular-biological methods known per se. Starting from the knownsubtilisin-producing microorganisms, it is also possible to isolatefurther natural subtilisin producers, to determine their subtilisinsequences and to develop them further, according to the guidelinesdevised herein. Bacterial species of this kind may also be cultured andused for appropriate production methods. Analogously, novel expressionvectors can be developed according to the model of the vectors disclosedin application WO 91/02792, for example. Cell-free expression systems inwhich protein biosynthesis is carried out in vitro may also beembodiments of the present invention, on the basis of the correspondingnucleic acid sequences. Any elements already set forth above may also becombined to give novel methods in order to prepare proteins of theinvention. In this connection, a multiplicity of possible combinationsof method steps for each protein of the invention is conceivable so thatthe optimal method should be determined experimentally for each specificindividual case.

Agents characterized in that they contain a proteolytic enzyme of theinvention are a separate subject matter of the invention.

Virtually all possible technical uses of enzymes of the invention dependon using the functional enzyme in an appropriate medium. Thus, forexample, the possible microbiological uses demand agents in which theenzyme, usually in the form of highly pure preparations, is combinedwith the necessary reaction partners or cofactors. Agents for thetreatment of raw materials or cosmetic preparations are likewisecharacterized by specific formulations. According to the invention, allthese formulations should be understood as being agents containing theenzyme of the invention.

Preferred embodiments included in this subject matter of the inventionare detergents or cleaning agents since, as the exemplary embodiments ofthe present application show, it was surprisingly found that asubtilisin variant 199I/211G (numbering according to B.lentus alkalineprotease) or a Bacillus lentus variant having the substitutionsS3T/V4I/V199I/L211G, in particular the B.lentus alkaline proteaseS3T/V4I/V199I/L211G variant derived from Bacillus lentus DSM 5483, givesa distinct performance increase on various soilings (compare Examples 2and 3), which exceeds the level of established detergent proteases withthe same amount of activity used. The same applies to correspondingmechanical dishwasher agents (compare Examples 4 and 5). This effectoccurs reproducibly both at different temperatures and at differentconcentrations.

This subject matter of the invention includes any conceivable types ofcleaning agents, both concentrates and agents to be applied in undilutedform; for use on the commercial scale, in the washing machine or formanual laundry or cleaning. They include, for example, detergents fortextiles, carpets or natural fibers, for which the term detergent isused in the present invention. They also include, for example,dishwashing agents for dishwashers or manual dishwashing agents orcleaners for hard surfaces such as metal, glass, porcelain, ceramic,tiles, stone, coated surfaces, plastics, wood or leather; for those, theterm cleaning agent is used in the present invention. Any type ofcleaning agent is an embodiment of the present invention, as long as aprotein of the invention has been added to it.

Embodiments of the present invention comprise any presentations of theagents of the invention, which are established in the prior art and/orappropriate. They include, for example, solid, pulverulent, liquid,gel-like or paste-like agents, where appropriate also composed of aplurality of phases, compressed or uncompressed; further examplesinclude: extrudates, granules, tablets or pouches, packaged both inlarge containers and in portions.

Agents of the invention contain enzymes of the invention in an amount offrom 2 μg to 20 mg and, increasingly preferably, from 5 μg to 17.5 mg,from 20 μg to 15 mg, from 50 μg to 10 mg, from 100 μg to 7.5 mg, from200 μg to 5 mg and from 500 μg to 1 mg, per gram of agent. This resultsin amounts of from 40 μg to 4 g and, increasingly preferably, from 50 μgto 3 g, from 100 μg to 2 g, from 200 μg to 1 g and, particularlypreferably, from 400 μg to 400 mg per application.

The protease activity in agents of this kind may be determined accordingto the method described in Tenside, Volume 7 (1970), pp. 125-132 and is,accordingly, indicated in protease units (PE=Protease-Einheiten). Theprotease activity of the agents may be up to 1,500,000 protease unitsper gram of preparation.

Apart from an enzyme important to the invention, an agent of theinvention contains, where appropriate, further ingredients such assurfactants, for example nonionic, anionic and/or amphotericsurfactants, and/or bleaches, and/or builders, and, where appropriate,further conventional ingredients.

The nonionic surfactants used are preferably alkoxylated, advantageouslyethoxylated, in particular primary alcohols having preferably from 8 to18 carbon atoms and, on average, from 1 to 12 mol of ethylene oxide (EO)per mole of alcohol, in which the alcohol radical can be linear or,preferably, methyl-branched in the 2-position or can comprise linear andmethyl-branched radicals in a mixture as are customarily present in oxoalcohol radicals. Particular preference is, however, given to alcoholethoxylates containing linear radicals of alcohols of native originhaving from 12 to 18 carbon atoms, for example from coconut, palm,tallow fatty or oleyl alcohol, and, on average, from 2 to 8 EO per moleof alcohol. Preferred ethoxylated alcohols include, for example,C₁₂₋₁₄-alcohols having 3 EO or 4 EO, C₉₋₁₁-alcohol having 7 EO,C₁₃₋₁₅-alcohols having 3 EO, 5 EO, 7 EO or 8 EO, C₁₂₋₁₈-alcohols having3 EO, 5 EO or 7 EO, and mixtures of these, such as mixtures ofC₁₂₋₁₄-alcohol having 3 EO and C₁₂₋₁₈-alcohol having 5 EO. The degreesof ethoxylation given are statistical averages which may be an integeror a fraction for a specific product. Preferred alcohol ethoxylates havea narrowed homolog distribution (narrow range ethoxylates, NRE). Inaddition to these nonionic surfactants, fatty alcohols having more than12 EO can also be used. Examples thereof are tallow fatty alcohol having14 EO, 25 EO, 30 EO or 40 EO.

A further class of preferably used nonionic surfactants which are usedeither as the sole nonionic surfactant or in combination with othernonionic surfactants are alkoxylated preferably ethoxylated orethoxylated and propoxylated fatty acid alkyl esters, preferably havingfrom 1 to 4 carbon atoms in the alkyl chain, in particular fatty acidmethyl esters.

A further class of nonionic surfactants which can advantageously be usedare the alkyl polyglycosides (APG). Alkyl polyglycosides which may beused satisfy the general formula RO(G)_(z), in which R is a linear orbranched, in particular methyl-branched in the 2-position, saturated orunsaturated, aliphatic radical having from 8 to 22, preferably from 12to 18 carbon atoms, and G is the symbol which stands for a glycose unithaving 5 or 6 carbon atoms, preferably for glucose. The degree ofglycosylation z is here between 1.0 and 4.0, preferably between 1.0 and2.0 and in particular between 1.1 and 1.4. Preference is given to usinglinear alkyl polyglucosides, i.e. alkyl polyglycosides in which thepolyglycosyl radical is a glucose radical, and the alkyl radical is ann-alkyl radical.

Nonionic surfactants of the amine oxide type, for exampleN-cocoalkyl-N,N-dimethylamine oxide and N-tallowalkyl-N,N-dihydroxyethylamine oxide, and of the fatty acid alkanolamidesmay also be suitable. The proportion of these nonionic surfactants ispreferably no more than that of the ethoxylated fatty alcohols, inparticular no more than half thereof.

Further suitable surfactants are polyhydroxy fatty acid amides of theformula (II)

in which RCO is an aliphatic acyl radical having from 6 to 22 carbonatoms, R¹ is hydrogen, an alkyl or hydroxyalkyl radical having from 1 to4 carbon atoms and [Z] is a linear or branched polyhydroxyalkyl radicalhaving from 3 to 10 carbon atoms and from 3 to 10 hydroxyl groups. Thepolyhydroxy fatty acid amides are known substances which can usually beobtained by reductive amination of a reducing sugar with ammonia, analkylamine or an alkanolamine and subsequent acylation with a fattyacid, a fatty acid alkyl ester or a fatty acid chloride.

The group of polyhydroxy fatty acid amides also includes compounds ofthe formula (III)

in which R is a linear or branched alkyl or alkenyl radical having from7 to 12 carbon atoms, R¹ is a linear, branched or cyclic alkyl radicalor an aryl radical having from 2 to 8 carbon atoms, and R² is a linear,branched or cyclic alkyl radical or an aryl radical or an oxy-alkylradical having from 1 to 8 carbon atoms, where C₁₋₄-alkyl or phenylradicals are preferred, and [Z] is a linear polyhydroxyalkyl radicalwhose alkyl chain is substituted with at least two hydroxyl groups, oralkoxylated, preferably ethoxylated or propoxylated, derivatives of thisradical.

[Z] is preferably obtained by reductive amination of a reducing sugar,for example glucose, fructose, maltose, lactose, galactose, mannose orxylose. The N-alkoxy- or N-aryloxy-substituted compounds may beconverted, for example by reaction with fatty acid methyl esters in thepresence of an alkoxide as catalyst, into the desired polyhydroxy fattyacid amides.

The anionic surfactants used are, for example, those of the sulfonateand sulfate type. Suitable surfactants of the sulfonate type arepreferably C₉₋₁₃-alkylbenzene sulfonates, olefin sulfonates, i.e.mixtures of alkene and hydroxyalkane sulfonates, and disulfonates, asobtained, for example, from C₁₂₋₁₈-monoolefins having a terminal orinternal double bond by sulfonation with gaseous sulfur trioxide andsubsequent alkaline or acidic hydrolysis of the sulfonation products.Also suitable are alkane sulfonates which are obtained fromC₁₂₋₁₈-alkanes, for example, by sulfochlorination or sulfoxidation withsubsequent hydrolysis or neutralization. Likewise suitable are also theesters of α-sulfo fatty acids (estersulfonates), for example theα-sulfonated methyl esters of hydrogenated coconut, palm kernel ortallow fatty acids.

Further suitable anionic surfactants are sulfated fatty acid glycerolesters. Fatty acid glycerol esters mean the mono-, di- and triesters,and mixtures thereof, as are obtained during the preparation byesterification of a monoglycerol with from 1 to 3 mol of fatty acid orduring the transesterification of triglycerides with from 0.3 to 2 molof glycerol. Preferred sulfated fatty acid glycerol esters are here thesulfation products of saturated fatty acids having from 6 to 22 carbonatoms, for example of capronic acid, caprylic acid, capric acid,myristic acid, lauric acid, palmitic acid, stearic acid or behenic acid.

Preferred alk(en)yl sulfates are the alkali metal, and in particular thesodium, salts of sulfuric half-esters of C₁₂-C₁₈-fatty alcohols, forexample of coconut fatty alcohol, tallow fatty alcohol, lauryl,myristyl, cetyl or stearyl alcohol or of C₁₀-C₂₀-oxo alcohols and thosehalf-esters of secondary alcohols of these chain lengths. Furtherpreferred are alk(en)yl sulfates of said chain length which comprise asynthetic, petrochemical-based straight-chain alkyl radical which haveanalogous degradation behavior to the equivalent compounds based onfatty chemical raw materials. From a washing performance viewpoint,preference is given to C₁₂-C₁₆-alkyl sulfates and C₁₂-C₁₅-alkylsulfates, and C₁₄-C₁₅-alkyl sulfates. 2,3-Alkyl sulfates are alsosuitable anionic surfactants.

The sulfuric monoesters of straight-chain or branched C₇₋₂₁-alcoholsethoxylated with from 1 to 6 mol of ethylene oxide, such as2-methyl-branched C₉₋₁₁-alcohols having, on average, 3.5 mol of ethyleneoxide (EO) or C₁₂₋₁₈-fatty alcohols having from 1 to 4 EO, are alsosuitable. Owing to their high foaming behavior, they are used incleaning agents only in relatively small amounts, for example in amountsup to 5% by weight, usually from 1 to 5% by weight.

Further suitable anionic surfactants are also the salts ofalkylsulfosuccinic acid, which are also referred to as sulfosuccinatesor as sulfosuccinic esters and which are monoesters and/or diesters ofsulfosuccinic acids with alcohols, preferably fatty alcohols and, inparticular, ethoxylated fatty alcohols. Preferred sulfosuccinatescontain C₈₋₁₈-fatty alcohol radicals or mixtures thereof. Particularlypreferred sulfosuccinates contain a fatty alcohol radical derived fromethoxylated fatty alcohols, which are themselves nonionic surfactants(see above for description). In this connection, sulfosuccinates whosefatty alcohol radicals are derived from ethoxylated fatty alcoholshaving a narrowed homolog distribution are, in turn, particularlypreferred. Likewise, it is also possible to use alk(en)ylsuccinic acidhaving preferably from 8 to 18 carbon atoms in the alk(en)yl chain orsalts thereof.

Further suitable anionic surfactants are, in particular, soaps.Saturated fatty acid soaps such as the salts of lauric acid, myristicacid, palmitic acid, stearic acid, hydrogenated erucic acid and behenicacid, and, in particular, soap mixtures derived from natural fattyacids, for example coconut, palm kernel or tallow fatty acids, aresuitable.

The anionic surfactants including soaps may be present in the form oftheir sodium, potassium or ammonium salts, and as soluble salts oforganic bases such as mono-, di- or triethanolamine. The anionicsurfactants are preferably in the form of their sodium or potassiumsalts, in particular in the form of the sodium salts.

The surfactants may be present in the cleaning agents or detergents ofthe invention in an overall amount of from preferably 5% by weight to50% by weight, in particular from 8% by weight to 30% by weight, basedon the finished agent.

Agents of the invention may contain bleaches. Of the compounds whichserve as bleaches and produce H₂O₂ in water, sodium percarbonate, sodiumperborate tetrahydrate and sodium perborate monohydrate are ofparticular importance. Other bleaches which can be used are, forexample, peroxopyrophosphates, citrate perhydrates and H₂O₂-producingperacidic salts or peracids, such as persulfates or persulfuric acid.Also useful is the urea peroxohydrate percarbamide which can bedescribed by the formula H₂N—CO—NH₂.H₂O₂. In particular when used forcleaning hard surfaces, for example for machine dishwashing, the agents,if desired, may also contain bleaches from the group of organicbleaches, although the use thereof is possible in principle also inagents for washing textiles. Typical organic bleaches are diacylperoxides such as, for example, dibenzoyl peroxide. Further typicalorganic bleaches are the peroxy acids, specific examples being alkylperoxy acids and aryl peroxy acids. Preferred representatives are peroxybenzoic acid and its ring-substituted derivatives, such asalkylperoxybenzoic acids, but also peroxy-α-naphthoic acid and magnesiummonoperphthalate, the aliphatic or substituted aliphatic peroxy acidssuch as peroxylauric acid, peroxystearic acid,ε-phthalimidoperoxycaproic acid (phthalimidoperoxyhexanoic acid, PAP),o-carboxy-benzamidoperoxycaproic acid, N-nonenylamidoperadipic acid andN-nonenylamidopersuccinate, and aliphatic and araliphaticperoxydicarboxylic acids such as 1,12-diperoxycarboxylic acid,1,9-diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid,diperoxyphthalic acids, 2-decyldiperoxybutane-1,4-dioic acid,N,N-terephthaloyl-di(6-aminopercaproic acid) may be used.

The bleach content of the agents may be from 1 to 40% by weight and, inparticular, from 10 to 20% by weight, using advantageously perboratemonohydrate or percarbonate.

In order to achieve improved bleaching action in cases of washing attemperatures of 60° C. and below, and in particular in the case oflaundry pretreatment, the agents may also include bleach activators.Bleach activators which can be used are compounds which, underperhydrolysis conditions, give aliphatic peroxocarboxylic acids havingpreferably from 1 to 10 carbon atoms, in particular from 2 to 4 carbonatoms, and/or substituted or unsubstituted perbenzoic acid. Substanceswhich carry O- and/or N-acyl groups of said number of carbon atomsand/or substituted or unsubstituted benzoyl groups are suitable.Preference is given to plurally acylated alkylenediamines, in particulartetraacetylethylenediamine (TAED), acylated triazine derivatives, inparticular 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT),acylated glycoluriles, in particular 1,3,4,6-tetraacetylglycoluril(TAGU), N-acylimides, in particular N-nonanoylsuccinimide (NOSI),acylated phenol sulfonates, in particular n-nonanoyl- orisononanoyloxybenzene sulfonate (n- or iso-NOBS), acylatedhydroxycarboxylic acids such as triethyl-O-acetyl citrate (TEOC),carboxylic anhydrides, in particular phthalic anhydride, isatoicanhydride and/or succinic anhydride, carboxamides such asN-methyldiacetamide, glycolide, acylated polyhydric alcohols, inparticular triacetin, ethylene glycol diacetate, isopropenyl acetate,2,5-diacetoxy-2,5-dihydrofuran and the enol esters disclosed in Germanpatent applications DE 196 16 693 and DE 196 16 767, and acetylatedsorbitol and mannitol, or mixtures thereof described in European patentapplication EP 0 525 239 (SORMAN), acylated sugar derivatives, inparticular pentaacetylglucose (PAG), pentaacetylfructose,tetraacetylxylose and octaacetyllactose, and acetylated, optionallyN-alkylated glucamine or gluconolactone, triazole or triazolederivatives and/or particulate caprolactams and/or caprolactamderivatives, preferably N-acylated lactams, for exampleN-benzoylcaprolactam and N-acetylcaprolactam, which are disclosed ininternational patent applications WO 94/27970, WO 94/28102, WO 94/28103,WO 95/00626, WO 95/14759 and WO 95/17498. The hydrophilicallysubstituted acyl acetals disclosed in German patent application DE 19616 769 and the acyl lactams described in German patent application DE196 16 770 and in international patent application WO 95/14075 arelikewise used with preference. It is also possible to use thecombinations of conventional bleach activators disclosed in Germanpatent application DE 44 43 177. Nitrile derivatives such ascyanopyridines, nitrile quats, e.g. N-alkylammoniumacetonitriles, and/orcyanamide derivatives may also be used. Preferred bleach activators aresodium 4-(octanoyloxy)benzenesulfonate, n-nonanoyl- orisononanoyloxybenzenesulfonate (n- or iso-NOBS),undecenoyloxybenzenesulfonate (UDOBS), sodiumdodecanoyloxybenzenesulfonate (DOBS), decanoyloxybenzoic acid (DOBA, OBC10) and/or dodecanoyloxybenzenesulfonate (OBS 12), andN-methylmorpholinium acetonitrile (MMA). Such bleach activators may bepresent in the customary quantitative range from 0.01 to 20% by weight,preferably in amounts from 0.1 to 15% by weight, in particular 1% byweight to 10% by weight, based on the total composition.

In addition to the conventional bleach activators or instead of them, itis also possible for “bleach catalysts” to be present. These substancesare bleach-enhancing transition metal salts or transition metalcomplexes such as, for example, Mn, Fe, Co, Ru or Mo salene complexes orcarbonyl complexes. Mn, Fe, Co, Ru, Mo, Ti, V and Cu complexescontaining N-containing tripod ligands, and Co, Fe, Cu and Ru amminecomplexes are also suitable as bleach catalysts, preference being givento using those compounds described in DE 197 09 284 A1. Acetonitrilederivatives, according to WO 99/63038, and bleach-activating transitionmetal complex compounds, according to WO 99/63041 are capable ofdeveloping a bleach-activating action in combination with amylases.

Agents of the invention usually contain one or more builders, inparticular zeolites, silicates, carbonates, organic cobuilders and,where no ecological reasons oppose their use, also phosphates. Thelatter are the preferred builders for use in particular in cleaningagents for machine dishwashing.

Compounds which may be mentioned here are crystalline, layered sodiumsilicates of the general formula NaMSi_(x)O_(2x+1).yH₂O, where M issodium or hydrogen, x is a number from 1.6 to 4, preferably from 1.9 to4.0, and y is a number from 0 to 20, and preferred values for x are 2, 3or 4. Crystalline phyllosilicates of this kind are described, forexample, in European patent application EP 0 164 514. Preferredcrystalline phyllosilicates of the formula indicated are those where Mis sodium and x adopts the values 2 or 3. In particular, both β- andδ-sodium disilicates Na₂Si₂O₅.yH₂O are preferred. Compounds of this kindare sold, for example, under the name SKS® (Clariant). Thus, SKS-6® isprimarily a δ-sodium disilicate having the formula Na₂Si₂O₅.yH₂O, andSKS-7® is primarily the β-sodium disilicate. Reacting the δ-sodiumdisilicate with acids (for example citric acid or carboxylic acid) giveskanemite NaHSi₂O₅.yH₂O, sold under the names SKS-9® and, respectively,SKS-10® (Clariant). It may also be advantageous to use chemicalmodifications of said phyllosilicates. The alkalinity of thephyllosilicates, for example, can thus be suitably influenced.Phyllosilicates doped with phosphate or with carbonate have, compared tothe δ-sodium disilicate, altered crystal morphologies, dissolve morerapidly and display an increased calcium binding ability, compared toδ-sodium disilicate. Thus, phyllosilicates of the general empiricalformula xNa₂O.ySiO₂O.zP₂O₅ where the x-to-y ratio corresponds to anumber from 0.35 to 0.6, the x-to-z ratio to a number from 1.75 to 1 200and the y-to-z ratio to a number from 4 to 2 800 are described in patentapplication DE 196 01 063. The solubility of the phyllosilicates mayalso be increased by using particularly finely granulatedphyllosilicates. It is also possible to use compounds of the crystallinephyllosilicates with other ingredients. Compounds which may be mentionedhere are in particular those with cellulose derivatives which haveadvantageous disintegrating action and are used in particular indetergent tablets, and those with polycarboxylates, for example citricacid, or polymeric polycarboxylates, for example copolymers of acrylicacid.

It is also possible to use amorphous sodium silicates having anNa₂O:SiO₂ modulus of from 1:2 to 1:3.3, preferably from 1:2 to 1:2.8,and in particular from 1:2 to 1:2.6, which have delayed dissolution andsecondary detergent properties. The dissolution delay relative toconventional amorphous sodium silicates can have been induced by variousmeans, for example by surface treatment, compounding,compaction/compression or by overdrying. Within the scope of thisinvention, the term “amorphous” also means “X-ray amorphous”. This meansthat in X-ray diffraction experiments the silicates do not give thesharp X-ray refractions typical of crystalline substances, but instead,at best, one or more maxima of these scattered X-rays, which have awidth of several degree units of the diffraction angle. However,particularly good builder properties will very likely result if, inelectron diffraction experiments, the silicate particles give poorlydefined or even sharp diffraction maxima. This is to be interpreted tothe effect that the products have microcrystalline regions with a sizefrom 10 to a few hundred nm, preference being given to values up to atmost 50 nm and in particular up to at most 20 nm. Particular preferenceis given to compressed/compacted amorphous silicates, compoundedamorphous silicates and overdried X-ray amorphous silicates.

A finely crystalline, synthetic zeolite containing bonded water, whichmay be used where appropriate, is preferably zeolite A and/or P. Aszeolite P, zeolite MAP® (commercial product from Crosfield) isparticularly preferred. However, zeolite X and mixtures of A, X and/or Pare also suitable. A product which is commercially available and can beused with preference within the scope of the present invention is, forexample, also a co-crystallizate of zeolite X and zeolite A (approx. 80%by weight zeolite X), which is sold by CONDEA Augusta S.p.A. under thetrade name VEGOBOND AX® and can be described by the formulanNa₂O.(1−n)K₂O.Al₂O₃.(2-2.5)SiO₂.(3.5-5.5)H₂O

Suitable zeolites have an average particle size of less than 10 μm(volume distribution; measurement method: Coulter counter) andpreferably contain from 18 to 22% by weight, in particular from 20 to22% by weight, of bonded water.

Use of the generally known phosphates as builder substances is of coursealso possible, provided such a use should not be avoided for ecologicalreasons. Among the multiplicity of commercially available phosphates,the alkali metal phosphates are the most important in the detergents andcleaning agents industry, with pentasodium or pentapotassiumtriphosphate (sodium or potassium tripolyphosphate) being particularlypreferred.

In this connection, alkali metal phosphates is the collective term forthe alkali metal (in particular sodium and potassium) salts of thevarious phosphoric acids, it being possible to differentiate betweenmetaphosphoric acids (HPO₃)_(n) and orthophosphoric acid H₃PO₄ as wellas higher molecular weight representatives. The phosphates combineseveral advantages: they act as alkali carriers, prevent lime depositson machine parts and lime incrustations in fabrics and, moreover,contribute to the cleaning performance.

Sodium dihydrogenphosphate, NaH₂PO₄, exists as dihydrate (density 1.91gcm⁻³, melting point 60° C.) and as monohydrate (density 2.04 gcm⁻³).Both salts are white powders which are very readily soluble in water andwhich lose their water of crystallization upon heating and at 200° C.convert to the weakly acidic diphosphate (disodium hydrogendiphosphate,Na₂H₂P₂O₇), at a higher temperature to sodium trimetaphosphate (Na₃P₃O₉)and Maddrell's salt (see below). NaH₂PO₄ is acidic; it forms whenphosphoric acid is adjusted to a pH of 4.5 using sodium hydroxidesolution and the suspension is sprayed. Potassium dihydrogenphosphate(primary or monobasic potassium phosphate, potassium biphosphate, KDP),KH₂PO₄, is a white salt of density 2.33 gcm⁻³, has a melting point of253° [decomposition with the formation of potassium polyphosphate(KPO₃)_(x)] and is readily soluble in water.

Disodium hydrogenphosphate (secondary sodium phosphate) Na₂HPO₄, is acolorless crystalline salt which is very readily soluble in water. Itexists in anhydrous form and with 2 mol (density 2.066 gcm⁻³, loss ofwater at 95° C.), 7 mol (density 1.68 gcm⁻³, melting point 48° C. withloss of 5 H₂O) and 12 mol (density 1.52 gcm⁻³, melting point 35° C. withloss of 5 H₂O) of water, becomes anhydrous at 100° C. and upon morevigorous heating converts to the diphosphate Na₄P₂O₇. Disodiumhydrogenphosphate is prepared by neutralizing phosphoric acid with sodasolution using phenolphthalein as indicator. Dipotassiumhydrogenphosphate (secondary or dibasic potassium phosphate), K₂HPO₄, isan amorphous, white salt which is readily soluble in water.

Trisodium phosphate, tertiary sodium phosphate, Na₃PO₄, are colorlesscrystals which, in the form of the dodecahydrate, have a density of 1.62gcm⁻³ and a melting point of 73-76° C. (decomposition), in the form ofthe decahydrate (corresponding to 19-20% P₂O₅) have a melting point of100° C. and in anhydrous form (corresponding to 39-40% P₂O₅) have adensity of 2.536 gcm⁻³. Trisodium phosphate is readily soluble in waterwith an alkaline reaction and is prepared by evaporating a solution ofexactly 1 mol of disodium phosphate and 1 mol of NaOH. Tripotassiumphosphate (tertiary or tribasic potassium phosphate), K₃PO₄, is a white,deliquescent granular powder of density 2.56 gcm⁻³, has a melting pointof 1 340° C. and is readily soluble in water with an alkaline reaction.It is produced, for example, during the heating of Thomas slag withcarbon and potassium sulfate. Despite the higher price, the more readilysoluble, and therefore highly effective, potassium phosphates are oftenpreferred over corresponding sodium compounds in the cleaning agentsindustry.

Tetrasodium diphosphate (sodium pyrophosphate), Na₄P₂O₇, exists inanhydrous form (density 2.534 gcm⁻³, melting point 988° C., also 880° C.given) and as decahydrate (density 1.815-1.836 gcm⁻³, melting point 94°C. with loss of water). Both substances are colorless crystals whichdissolve in water with an alkaline reaction. Na₄P₂O₇ is formed duringthe heating of disodium phosphate to >200° C. or by reacting phosphoricacid with soda in a stoichiometric ratio and dewatering the solution byspraying. The decahydrate complexes heavy metal salts and hardnessconstituents and thus reduces the water hardness. Potassium diphosphate(potassium pyrophosphate), K₄P₂O₇, exists in the form of the trihydrateand is a colorless, hygroscopic powder of density 2.33 gcm⁻³, which issoluble in water, the pH of the 1% strength solution at 25° C. being10.4.

Condensation of NaH₂PO₄ and KH₂PO₄ results in higher molecular weightsodium phosphates and potassium phosphates, respectively, amongst whichcyclic representatives, the sodium and potassium metaphosphates,respectively, and chain-shaped types, the sodium and potassiumpolyphosphates, respectively, can be differentiated. Particularly forthe latter, a multiplicity of names are in use: melt or thermalphosphates, Graham's salt, Kurrol's and Maddrell's salt. All highersodium and potassium phosphates are together referred to as condensedphosphates.

The industrially important pentasodium triphosphate, Na₅P₃O₁₀ (sodiumtripolyphosphate), is a nonhygroscopic, white, water-soluble salt whichis anhydrous or crystallizes with 6 H₂O and is of the general formulaNaO-[P(O)(ONa)-O]_(n)-Na where n=3. In 100 g of water, about 17 g of thesalt which is free of water of crystallization dissolve at roomtemperature, approx. 20 g dissolve at 60° C., and about 32 g dissolve at100° C.; if the solution is heated at 100° C. for two hours, about 8% oforthophosphate and 15% of diphosphate form due to hydrolysis. In thepreparation of pentasodium triphosphate, phosphoric acid is reacted withsoda solution or sodium hydroxide solution in a stoichiometric ratio,and the solution is dewatered by spraying. Similarly to Graham's saltand sodium diphosphate, pentasodium triphosphate dissolves manyinsoluble metal compounds (including lime soaps, etc.). Pentapotassiumtriphosphate, K₅P₃O₁₀ (potassium tripolyphosphate), is availablecommercially, for example, in the form of a 50% strength by weightsolution (>23% P₂O₅, 25% K₂O). The potassium polyphosphates are usedwidely in the detergents and cleaning agents industry. In addition,sodium potassium tripolyphosphates also exist which can likewise be usedwithin the scope of the present invention. These form, for example, whensodium trimetaphosphate is hydrolyzed with KOH:(NaPO₃)₃+2 KOH→Na₃K₂P₃O₁₀+H₂O

According to the invention, these can be used exactly as sodiumtripolyphosphate, potassium tripolyphosphate or mixtures of these two;mixtures of sodium tripolyphosphate and sodium potassiumtripolyphosphate or mixtures of potassium tripolyphosphate and sodiumpotassium tripolyphosphate or mixtures of sodium tripolyphosphate andpotassium tripolyphosphate and sodium potassium tripolyphosphate canalso be used according to the invention.

Organic cobuilders which can be used in the detergents and cleaningagents of the invention are, in particular, polycarboxylates orpolycarboxylic acids, polymeric polycarboxylates, polyaspartic acid,polyacetals, optionally oxidized dextrins, further organic cobuilders(see below), and phosphonates. These classes of substance are describedbelow.

Useable organic builder substances are, for example, the polycarboxylicacids usable in the form of their sodium salts, the term polycarboxylicacids meaning those carboxylic acids which carry more than one acidfunction. Examples of these are citric acid, adipic acid, succinic acid,glutaric acid, malic acid, tartaric acid, maleic acid, fumaric acid,sugar acids, aminocarboxylic acids, nitrilotriacetic acids (NTA), aslong as such a use should not be avoided for ecological reasons, andmixtures thereof. Preferred salts are the salts of the polycarboxylicacids such as citric acid, adipic acid, succinic acid, glutaric acid,tartaric acid, sugar acids, and mixtures thereof.

It is also possible to use the acids per se. In addition to theirbuilder action, the acids typically also have the property of anacidifying component and thus also serve to establish a lower and milderpH of detergents or cleaning agents, as long as the pH resulting fromthe mixture of the remaining components is not desired. Particularmention should be made here of environmentally safe acids such as citricacid, acetic acid, tartaric acid, malic acid, lactic acid, glycolicacid, succinic acid, glutaric acid, adipic acid, gluconic acid and anymixtures thereof. However, mineral acids, in particular sulfuric acid,or bases, in particular ammonium or alkali metal hydroxides, may alsoserve as pH regulators. The agents of the invention contain suchregulators in amounts of preferably not more than 20% by weight, inparticular from 1.2% by weight to 17% by weight.

Suitable builders are also polymeric polycarboxylates; these are, forexample, the alkali metal salts of polyacrylic acid or ofpolymethacrylic acid, for example those having a relative molecular massof from 500 to 70 000 g/mol.

The molar masses given for polymeric polycarboxylates are, for thepurposes of this specification, weight-average molar masses, M_(W), ofthe respective acid form, determined in principle by means of gelpermeation chromatography (GPC), using a UV detector. The measurementwas made against an external polyacrylic acid standard which, owing toits structural similarity toward the polymers studied, providesrealistic molecular weight values. These figures differ considerablyfrom the molecular weight values obtained using polystyrenesulfonicacids as the standard. The molar masses measured againstpolystyrenesulfonic acids are usually considerably higher than the molarmasses given in this specification.

Suitable polymers are, in particular, polyacrylates which preferablyhave a molecular mass of from 2 000 to 20 000 g/mol. Owing to theirsuperior solubility, preference in this group may be given in turn tothe short-chain polyacrylates which have molar masses of from 2 000 to10 000 g/mol, and particularly preferably from 3 000 to 5 000 g/mol.

Also suitable are copolymeric polycarboxylates, in particular those ofacrylic acid with methacrylic acid and of acrylic acid or methacrylicacid with maleic acid. Copolymers which have proven to be particularlysuitable are those of acrylic acid with maleic acid which contain from50 to 90% by weight of acrylic acid and from 50 to 10% by weight ofmaleic acid. Their relative molecular mass, based on free acids, isgenerally from 2 000 to 70 000 g/mol, preferably 20 000 to 50 000 g/moland in particular 30 000 to 40 000 g/mol. The (co)polymericpolycarboxylates may be used either as powders or as aqueous solution.The (co)polymeric polycarboxylates may be from 0.5 to 20% by weight, inparticular 1 to 10% by weight of the content of the agent.

To improve the solubility in water, the polymers may also containallylsulfonic acids such as, for example, allyloxybenzenesulfonic acidand methallylsulfonic acid as monomers.

Particular preference is also given to biodegradable polymers of morethan two different monomer units, for example those which contain, asmonomers, salts of acrylic acid and of maleic acid, and vinyl alcohol orvinyl alcohol derivatives, or those which contain, as monomers, salts ofacrylic acid and of 2-alkylallylsulfonic acid, and sugar derivatives.

Further preferred copolymers are those which preferably have, asmonomers, acrolein and acrylic acid/acrylic acid salts or acrolein andvinyl acetate.

Further preferred builder substances which may be mentioned are alsopolymeric aminodicarboxylic acids, their salts or their precursorsubstances. Particular preference is given to polyaspartic acids orsalts and derivatives thereof.

Further suitable builder substances are polyacetals which can beobtained by reacting dialdehydes with polyolcarboxylic acids having from5 to 7 carbon atoms and at least 3 hydroxyl groups. Preferredpolyacetals are obtained from dialdehydes such as glyoxal,glutaraldehyde, terephthalaldehyde and mixtures thereof and frompolyolcarboxylic acids such as gluconic acid and/or glucoheptonic acid.

Further suitable organic builder substances are dextrins, for exampleoligomers or polymers of carbohydrates, which can be obtained by partialhydrolysis of starches. The hydrolysis can be carried out by customaryprocesses, for example acid-catalyzed or enzyme-catalyzed processes. Thehydrolysis products preferably have average molar masses in the rangefrom 400 to 500 000 g/mol. Preference is given here to a polysaccharidehaving a dextrose equivalent (DE) in the range from 0.5 to 40, inparticular from 2 to 30, where DE is a common measure of the reducingaction of a polysaccharide compared with dextrose which has a DE of 100.It is possible to use both maltodextrins having a DE between 3 and 20and dried glucose syrups having a DE between 20 and 37, and also “yellowdextrins” and “white dextrins” with higher molar masses in the rangefrom 2 000 to 30 000 g/mol.

The oxidized derivatives of such dextrins are their reaction productswith oxidation agents which are able to oxidize at least one alcoholfunction of the saccharide ring to the carboxylic acid function.Particularly preferred organic builders for agents of the invention areoxidized starches and derivatives thereof of the applications EP 472042,WO 97/25399 and EP 755944, respectively.

Oxydisuccinates and other derivatives of disuccinates, preferablyethylenediamine disuccinate, are also further suitable cobuilders. Here,ethylenediamine N,N′-disuccinate (EDDS) is preferably used in the formof its sodium or magnesium salts. In this connection, further preferenceis also given to glycerol disuccinates and glycerol trisuccinates.Suitable use amounts in zeolite-containing, carbonate-containing and/orsilicate-containing formulations are between 3 and 15% by weight.

Further organic cobuilders which may be used are, for example,acetylated hydroxycarboxylic acids or salts thereof, which may also bepresent, where appropriate, in lactone form and which contain at least 4carbon atoms and at least one hydroxy group and at most two acid groups.

A further class of substance having cobuilder properties is thephosphonates. These are, in particular, hydroxyalkane and aminoalkanephosphonates. Among the hydroxyalkane phosphonates, 1-hydroxyethane1,1-diphosphonate (HEDP) is of particular importance as a cobuilder. Itis preferably used as sodium salt, the disodium salt being neutral andthe tetrasodium salt being alkaline (pH 9). Suitable aminoalkanephosphonates are preferably ethylenediaminetetra-methylene phosphonate(EDTMP), diethylenetriamine-pentamethylene phosphonate (DTPMP) andhigher homologs thereof. They are preferably used in the form of theneutral sodium salts, for example as the hexasodium salt of EDTMP or asthe hepta- and octasodium salt of DTPMP. Here, preference is given tousing HEDP as builder from the class of phosphonates. In addition, theaminoalkane phosphonates have a marked heavy metal-binding capacity.Accordingly, particularly if the agents also contain bleaches, it may bepreferable to use aminoalkane phosphonates, in particular DTPMP, ormixtures of said phosphonates.

In addition, all compounds which are able to form complexes withalkaline earth metal ions can be used as cobuilders.

The agents of the invention may contain builder substances, whereappropriate, in amounts of up to 90% by weight, and preferably containthem in amounts of up to 75% by weight. Detergents of the invention havebuilder contents of, in particular, from 5% by weight to 50% by weight.In inventive agents for cleaning hard surfaces, in particular formachine cleaning of dishes, the builder substance content is inparticular from 5% by weight to 88% by weight, with preferably nowater-insoluble builder materials being used in such agents. A preferredembodiment of inventive agents for, in particular, machine cleaning ofdishes contains from 20% by weight to 40% by weight water-solubleorganic builders, in particular alkali metal citrate, from 5% by weightto 15% by weight alkali metal carbonate and from 20% by weight to 40% byweight alkali metal disilicate.

Solvents which may be used in the liquid to gelatinous compositions ofdetergents and cleaning agents are, for example, from the group ofmonohydric or polyhydric alcohols, alkanolamines or glycol ethers, aslong as they are miscible with water in the given concentration range.Preferably, the solvents are selected from ethanol, n- or i-propanol,butanols, ethylene glycol methyl ether, ethylene glycol ethyl ether,ethylene glycol propyl ether, ethylene glycol mono-n-butyl ether,diethylene glycol methyl ether, diethylene glycol ethyl ether, propyleneglycol methyl, ethyl or propyl ether, dipropylene glycol monomethyl ormonoethyl ether, diisopropylene glycol monomethyl or monoethyl ether,methoxy, ethoxy or butoxy triglycol, 1-butoxyethoxy-2-propanol,3-methyl-3-methoxybutanol, propylene glycol t-butyl ether, and mixturesof these solvents.

Solvents may be used in the liquid to gelatinous detergents and cleaningagents of the invention in amounts of between 0.1 and 20% by weight, butpreferably below 15% by weight, and in particular below 10% by weight.

To adjust the viscosity, one or more thickeners or thickening systemsmay be added to the composition of the invention. These high molecularweight substances which are also called swell(ing) agents usually soakup the liquids and swell in the process, converting ultimately intoviscous true or colloidal solutions.

Suitable thickeners are inorganic or polymeric organic compounds.Inorganic thickeners include, for example, polysilicic acids, clayminerals, such as montmorillonites, zeolites, silicas and bentonites.The organic thickeners are from the groups of natural polymers, modifiednatural polymers and completely synthetic polymers. Such naturalpolymers are, for example, agar-agar, carrageen, tragacanth, gum arabic,alginates, pectins, polyoses, guar flour, carob seed flour, starch,dextrins, gelatins and casein. Modified natural substances which areused as thickeners are primarily from the group of modified starches andcelluloses. Examples which may be mentioned here arecarboxymethylcellulose and other cellulose ethers, hydroxyethylcelluloseand hydroxypropylcellulose, and carob flour ether. Completely syntheticthickeners are polymers such as polyacrylic and polymethacryliccompounds, vinyl polymers, polycarboxylic acids, polyethers, polyimines,polyamides and polyurethanes.

The thickeners may be present in an amount up to 5% by weight,preferably from 0.05 to 2% by weight, and particularly preferably from0.1 to 1.5% by weight, based on the finished composition.

The detergent and cleaning agent of the invention may, whereappropriate, comprise, as further customary ingredients, sequesteringagents, electrolytes and further excipients such as optical brighteners,graying inhibitors, silver corrosion inhibitors, color transferinhibitors, foam inhibitors, abrasive substances, dyes and/orfragrances, and microbial active substances and/or UV-absorbing agents.

The textile detergents of the invention may contain, as opticalbrighteners, derivatives of diaminostilbene-disulfonic acid or alkalimetal salts thereof. Suitable are, for example, salts of4,4′-bis(2-anilino-4-morpholino-1,3,5-triazinyl-6-amino)stilbene-2,2′-disulfonicacid or similarly constructed compounds which carry a diethanolaminogroup, a methylamino group, an anilino group or a 2-methoxyethylaminogroup instead of the morpholino group. In addition, brighteners of thesubstituted diphenylstyryl type may be present, for example the alkalimetal salts of 4,4′-bis(2-sulfostyryl)diphenyl,4,4′-bis(4-chloro-3-sulfostyryl)diphenyl, or4-(4-chlorostyryl)-4′-(2-sulfostyryl)diphenyl. Mixtures of theabove-mentioned optical brighteners may also be used.

Graying inhibitors have the function of keeping the soil detached fromthe textile fiber in suspension in the liquor. Suitable for this purposeare water-soluble colloids, usually organic in nature, for examplestarch, glue, gelatin, salts of ethercarboxylic acids or ethersulfonicacids of starch or of cellulose, or salts of acidic sulfuric esters ofcellulose or of starch. Water-soluble polyamides containing acidicgroups are also suitable for this purpose. Furthermore, starchderivatives other than those mentioned above may be used, for examplealdehyde starches. Preference is given to cellulose ethers such ascarboxymethyl-cellulose (Na salt), methylcellulose,hydroxyalkylcellulose and mixed ethers such asmethylhydroxyethylcellulose, methylhydroxypropylcellulose,methylcarboxymethylcellulose, and mixtures thereof, for example inamounts of from 0.1 to 5% by weight, based on the agents.

In order to protect against silver corrosion, silver corrosioninhibitors may be used in dishwashing cleaning agents of the invention.Such inhibitors are known in the prior art, for example benzotriazoles,iron(III) chloride or CoSO₄. As, for example, European patent EP 0 736084 B1 discloses, silver corrosion inhibitors which are particularlysuitable for being used together with enzymes are manganese, titanium,zirconium, hafnium, vanadium, cobalt, or cerium salts and/or complexesin which the specified metals are present in any of the oxidation stagesII, III, IV, V or VI. Examples of such compounds are MnSO₄, V₂O₅, V₂O₄,VO₂, TiOSO₄, K₂TiF₆, K₂ZrF₆, Co(NO₃)₂, Co(NO₃)₃, and mixtures thereof.

Soil-release active ingredients or soil repellents are usually polymerswhich, when used in a detergent, impart soil-repellent properties to thelaundry fiber and/or assist the ability of the other detergentingredients to detach soil. A comparable effect can also be observedwith their use in cleaning agents for hard surfaces.

Soil-release active ingredients which are particularly effective andhave been known for a long time are copolyesters having dicarboxylicacid, alkylene glycol and polyalkylene glycol units. Examples thereofare copolymers or mixed polymers of polyethylene terephthalate andpolyoxyethylene glycol (DT 16 17 141, and, respectively, DT 22 00 911).German Offenlegungs-schrift DT 22 53 063 discloses acidic agentscontaining, inter alia, a copolymer of a dibasic carboxylic acid and analkylene or cycloalkylene polyglycol. German documents DE 28 57 292 andDE 33 24 258 and European patent EP 0 253 567 describe polymers ofethylene terephthalate and polyethylene oxide terephthalate and the usethereof in detergents. European patent EP 066 944 relates to agentscontaining a copolyester of ethylene glycol, polyethylene glycol,aromatic dicarboxylic acid and sulfonated aromatic dicarboxylic acid inparticular molar ratios. European patent EP 0 185 427 discloses methylor ethyl group end-group-capped polyesters having ethylene and/orpropylene terephthalate and polyethylene oxide terephthalate units, anddetergents containing such a soil-release polymer. European patent EP 0241 984 discloses a polyester which contains, in addition to oxyethylenegroups and terephthalic acid units also substituted ethylene units andglycerol units. European patent EP 0 241 985 discloses polyesters whichcontain, in addition to oxyethylene groups and terephthalic acid units,1,2-propylene, 1,2-butylene and/or 3-methoxy-1,2-propylene groups, andglycerol units and which are end-group-capped with C₁- to C₄-alkylgroups. European patent application EP 0 272 033 discloses polyestershaving polypropylene terephthalate and polyoxyethylene terephthalateunits, which are at least partially end-group-capped by C₁₋₄-alkyl oracyl radicals. European patent EP 0 274 907 describes sulfoethylend-group-capped terephthalate-containing soil-release polyesters.According to European patent application EP 0 357 280, sulfonation ofunsaturated end groups produces soil-release polyesters havingterephthalate, alkylene glycol and poly-C₂₋₄-glycol units. Internationalpatent application WO 95/32232 relates to acidic, aromatic polyesterscapable of detaching soil. International patent application WO 97/31085discloses nonpolymeric soil-repellent active ingredients for materialsmade of cotton, which have a plurality of functional units: a first unitwhich may be cationic, for example, is able to adsorb to the cottonsurface by means of electrostatic interaction, and a second unit whichis hydrophobic is responsible for the active ingredient remaining at thewater/cotton interface.

The color transfer inhibitors suitable for use in laundry detergents ofthe invention include, in particular, polyvinylpyrrolidones,polyvinylimidazoles, polymeric N-oxides such as poly(vinylpyridineN-oxide) and copolymers of vinylpyrrolidone with vinylimidazole.

For use in machine cleaning processes, it may be of advantage to addfoam inhibitors to the agents. Examples of suitable foam inhibitors aresoaps of natural or synthetic origin having a high proportion of C₁₈-C₂₄fatty acids. Examples of suitable nonsurfactant-type foam inhibitors areorganopolysiloxanes and their mixtures with microfine, optionallysilanized silica and also paraffins, waxes, microcrystalline waxes, andmixtures thereof with silanized silica or bis-stearyl-ethylenediamide.With advantages, use is also made of mixtures of different foaminhibitors, for example mixtures of silicones, paraffins or waxes. Thefoam inhibitors, in particular those containing silicone and/orparaffin, are preferably bound to a granular, water-soluble ordispersible support substance. Particular preference is given here tomixtures of paraffins and bis-stearylethylenediamides.

A cleaning agent of the invention for hard surfaces may, in addition,contain ingredients with abrasive action, in particular from the groupcomprising quartz flours, wood flours, polymer flours, chalks and glassmicrobeads, and mixtures thereof. Abrasives are present in the cleaningagents of the invention preferably at not more than 20% by weight, inparticular from 5% by weight to 15% by weight.

Dyes and fragrances are added to detergents and cleaning agents in orderto improve the esthetic appeal of the products and to provide theconsumer, in addition to washing and cleaning performance, with avisually and sensorially “typical and unmistakable” product. As perfumeoils and/or fragrances it is possible to use individual odorantcompounds, for example the synthetic products of the ester, ether,aldehyde, ketone, alcohol and hydrocarbon types. Odorant compounds ofthe ester type are, for example, benzyl acetate, phenoxyethylisobutyrate, p-tert-butylcyclohexyl acetate, linalyl acetate,dimethylbenzylcarbinyl acetate, phenylethyl acetate, linalyl benzoate,benzyl formate, ethyl methylphenyl glycinate, allylcyclohexylpropionate, styrallyl propionate and benzyl salicylate. The ethersinclude, for example, benzyl ethyl ether; the aldehydes include, forexample, the linear alkanals having 8-18 carbon atoms, citral,citronellal, citronellyloxyacetaldehyde, cyclamenaldehyde,hydroxycitronellal, lilial and bourgeonal; the ketones include, forexample, the ionones, α-isomethylionone and methyl cedryl ketone; thealcohols include anethol, citronellol, eugenol, geraniol, linalool,phenylethyl alcohol, and terpineol; the hydrocarbons include primarilythe terpenes such as limonene and pinene. Preference, however, is givento the use of mixtures of different odorants which together produce anappealing fragrance note. Such perfume oils may also contain naturalorodant mixtures, as obtainable from plant sources, for example pineoil, citrus oil, jasmine oil, patchouli oil, rose oil or ylang-ylangoil. Likewise suitable are muscatel, sage oil, camomile oil, clove oil,balm oil, mint oil, cinnamon leaf oil, lime blossom oil, juniper berryoil, vetiver oil, olibanum oil, galbanum oil and labdanum oil, and alsoorange blossom oil, neroli oil, orangepeel oil and sandalwood oil. Thedye content of detergents and cleaning agents is usually less than 0.01%by weight, while fragrances may make up up to 2% by weight of theoverall formulation.

The fragrances may be incorporated directly into the detergents andcleaning agents; however, it may also be advantageous to apply thefragrances to carriers which intensify the adhesion of the perfume tothe material to be cleaned and, by means of slower fragrance release,ensure long-lasting fragrance, in particular of treated textiles.Materials which have become established as such carriers are, forexample, cyclodextrins, it being possible, in addition, for thecyclodextrin-perfume complexes to be additionally coated with furtherauxiliaries. Another preferred carrier for fragances is the describedzeolite X which can also absorb fragrances instead of or in a mixturewith surfactants. Preference is therefore given to detergents andcleaning agents which contain the described zeolite X and fragranceswhich, preferably, are at least partially absorbed on the zeolite.

Preferred dyes whose selection is by no means difficult for the skilledworker have high storage stability and insensitivity to the otheringredients of the agents and to light, and also have no pronouncedaffinity for textile fibers, so as not to stain them.

To control microorganisms, detergents or cleaning agents may containantimicrobial active ingredients. Depending on antimicrobial spectrumand mechanism of action, a distinction is made here betweenbacteriostatics and bactericides, fungistatics and fungicides, etc.Examples of important substances from these groups are benzalkoniumchlorides, alkylaryl sulfonates, halogen phenols and phenol mercuryacetate. The terms antimicrobial action and antimicrobial activeingredient have, within the teaching of the invention, the meaningcommon in the art, which is described, for example, by K. H. Wallhäuβerin “Praxis der Sterilisation, Desinfektion—Konservierung:Keimidentifizierung—Betriebshygiene” (5th Edition,—Stuttgart; New York:Thieme, 1995), it being possible to use all of the substances havingantimicrobial action described there. Suitable antimicrobial activeingredients are preferably selected from the groups of alcohols, amines,aldehydes, antimicrobial acids or their salts, carboxylic esters, acidamides, phenols, phenol derivatives, diphenyls, diphenylalkanes, ureaderivatives, oxygen acetals, nitrogen acetals and also oxygen andnitrogen formals, benzamidines, isothioazolines, phthalimidederivatives, pyridine derivatives, antimicrobial surfactant compounds,guanidines, antimicrobial amphoteric compounds, quinolines,1,2-dibromo-2,4-dicyanobutane, iodo-2-propylbutyl carbamate, iodine,iodophors, peroxo compounds, halogen compounds, and any mixtures of theabove.

The antimicrobial active ingredient may be selected from ethanol,n-propanol, isopropanol, 1,3-butanediol, phenoxyethanol, 1,2-propyleneglycol, glycerol, undecylenic acid, benzoic acid, salicylic acid,dihydracetic acid, o-phenylphenol, N-methylmorpholino-acetonitrile(MMA), 2-benzyl-4-chlorophenol,2,2′-methylenebis(6-bromo-4-chlorophenol),4,4′-dichloro-2′-hydroxydiphenyl ether (dichlosan),2,4,4′-trichloro-2′-hydroxydiphenyl ether (trichlosan), chlorohexidine,N-(4-chlorophenyl)-N-(3,4-dichlorophenyl)urea,N,N′-(1,10-decanediyldi-1-pyridinyl-4-ylidene)-bis(1-octanamine)dihydrochloride,N,N′-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetra-azatetradecanediimidamide,glucoprotamines, antimicrobial surface-active quaternary compounds,guanidines including the bi- and polyguanidines, such as, for example,1,6-bis(2-ethylhexylbiguanidohexane) dihydrochloride,1,6-di-(N₁,N_(l)′-phenyldiguanido-N₅,N₅′)hexane tetrahydrochloride,1,6-di-(N₁,N₁′-phenyl-N₁,N₁-methyldiguanido-N₅,N₅′)hexanedihydrochloride, 1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)hexanedihydrochloride,1,6-di-(N₁,N₁′-2,6-dichlorophenyl-diguanido-N₅,N₅′)hexanedihydrochloride,1,6-di-[N₁,N₁′-beta-(p-methoxyphenyl)diguanido-N₅,N₅′]hexanedihydrochloride,1,6-di-(N₁,N₁′-alpha-methyl-beta-phenyldiguanido-N₅,N₅′)hexanedihydrochloride, 1,6-di-(N₁,N₁′-p-nitrophenyldiguanido-N₅,N₅′)hexanedihydrochloride,omega:omega-di-(N₁,N_(l)′-phenyldiguanido-N₅,N₅′)-di-n-propyl etherdihydrochloride,omega:omega′-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)-di-n-propylether tetrahydrochloride,1,6-di-(N₁,N₁′-2,4-dichlorophenyldiguanido-N₅,N₅′)hexanetetrahydrochloride, 1,6-di-(N₁,N₁′-p-methylphenyldiguanido-N₅,N₅′)hexanedihydrochloride,1,6-di-(N₁,N₁′-2,4,5-trichlorophenyldiguanido-N₅,N₅′)hexanetetrahydrochloride,1,6-di-[N₁,N₁′-alpha-(p-chlorophenyl)ethyldiguanido-N₅,N₅′]-hexanedihydrochloride, omega:omega-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)m-xylenedihydrochloride, 1,12-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)dodecanedihydrochloride, 1,10-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)decanetetrahydrochloride, 1,12-di-(N₁,N₁′-phenyldiguanido-N5,N₅′)dodecanetetrahydrochloride, 1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)hexanedihydrochloride, 1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)hexanetetrahydrochloride, ethylene-bis(1-tolylbiguanide),ethylene-bis(p-tolylbiguanide),ethylene-bis(3,5-dimethylphenylbiguanide),ethylene-bis(p-tert-amylphenylbiguanide),ethylene-bis(nonyl-phenylbiguanide), ethylene-bis(phenylbiguanide),ethylene-bis(N-butylphenylbiguanide),ethylene-bis(2,5-diethoxyphenylbiguanide),ethylene-bis(2,4-dimethylphenylbiguanide),ethylene-bis(o-diphenylbiguanide), ethylene-bis(mixed amylnaphthylbiguanide), N-butylethylene-bis(phenylbiguanide),trimethylenebis(o-tolylbiguanide),N-butyl-trimethyl-bis(phenylbiguanide) and the corresponding salts suchas acetates, gluconates, hydrochlorides, hydrobromides, citrates,bisulfites, fluorides, polymaleates, N-cocoalkyl sarcosinates,phosphites, hypophosphites, perfluorooctanoates, silicates, sorbates,salicylates, maleates, tartrates, fumarates,ethylenediaminetetraacetates, iminodiacetates, cinnamates, thiocyanates,arginates, pyromellitates, tetracarboxybutyrates, benzoates, glutarates,monofluorophosphates, perfluoropropionates, and any mixtures thereof.Also suitable are halogenated xylene and cresol derivatives, such asp-chlorometacresol or p-chlorometaxylene, and natural antimicrobialactive ingredients of plant origin (for example from spices or herbs),animal origin and microbial origin. Preference may be given to usingantimicrobial surface-active quaternary compounds, a naturalantimicrobial active ingredient of plant origin and/or a naturalantimicrobial active ingredient of animal origin, most preferably atleast one natural antimicrobial active ingredient of plant origin fromthe group comprising caffeine, theobromine and theophylline andessential oils such as eugenol, thymol and geraniol, and/or at least onenatural antimicrobial active ingredient of animal origin from the groupcomprising enzymes such as milk protein, lysozyme and lactoperoxidase,and/or at least one antimicrobial surface-active quaternary compoundhaving an ammonium, sulfonium, phosphonium, iodonium or arsonium group,peroxo compounds and chlorine compounds. It is also possible to usesubstances of microbial origin, the “bacteriocines”.

The quaternary ammonium compounds (QACs) which are suitable asantimicrobial active ingredients have the general formula (R¹) (R²) (R³)(R⁴) N⁺ X⁻ where R¹ to R⁴ are identical or different C₁-C₂₂-alkylradicals, C₇-C₂₈-aralkyl radicals or heterocyclic radicals, where two,or in the case of an aromatic incorporation as in pyridine, even threeradicals, together with the nitrogen atom, form the heterocycle, forexample a pyridinium or imidazolinium compound, and X⁻ are halide ions,sulfate ions, hydroxide ions or similar anions. For optimalantimicrobial action, at least one of the radicals preferably has achain length of from 8 to 18, in particular 12 to 16, carbon atoms.

QACs can be prepared by reacting tertiary amines with alkylating agentssuch as, for example, methyl chloride, benzyl chloride, dimethylsulfate, dodecyl bromide, or else ethylene oxide. The alkylation oftertiary amines having one long alkyl radical and two methyl groupsproceeds particularly readily, and the quaternization of tertiary amineshaving two long radicals and one methyl group can also be carried outwith the aid of methyl chloride under mild conditions. Amines which havethree long alkyl radicals or hydroxy-substituted alkyl radicals have lowreactivity and are preferably quaternized using dimethyl sulfate.

Examples of suitable QACs are benzalkonium chloride(N-alkyl-N,N-dimethylbenzylammonium chloride, CAS No. 8001-54-5),benzalkone B (m,p-dichlorobenzyldimethyl-C12-alkylammonium chloride, CASNo. 58390-78-6), benzoxonium chloride(benzyldodecyl-bis(2-hydroxyethyl)ammonium chloride), cetrimoniumbromide (N-hexadecyl-N,N-trimethylammonium bromide, CAS No. 57-09-0),benzetonium chloride(N,N-dimethyl-N-[2-[2-[p-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethyl]-benzylammoniumchloride, CAS No. 121-54-0), dialkyldimethylammonium chlorides such asdi-n-decyldimethylammonium chloride (CAS No. 7173-51-5-5),didecyldimethylammonium bromide (CAS No. 2390-68-3),dioctyldimethylammonium chloride, 1-cetylpyridinium chloride (CAS No.123-03-5) and thiazoline iodide (CAS No. 15764-48-1), and mixturesthereof. Particularly preferred QACs are the benzalkonium chlorideshaving C₈-C₁₈-alkyl radials, in particularC₁₂-C₁₄-alkyl-benzyldimethylammonium chloride.

Benzalkonium halides and/or substituted benzalkonium halides arecommercially available, for example, as Barquat® ex Lonza, Marquat® exMason, Variquat® ex Witco/Sherex and Hyamine® ex Lonza, and Bardac® exLonza. Further commercially available antimicrobial active ingredientsare N-(3-chloroallyl)hexaminium chloride such as Dowicide® and Dowicil®ex Dow, benzethonium chloride such as Hyamine® 1622 ex Rohm & Haas,methylbenzethonium chloride such as Hyamine® 10X ex Rohm & Haas,cetylpyridinium chloride such as cepacol chloride ex Merrell Labs.

The antimicrobial active ingredients are used in amounts of from 0.0001%by weight to 1% by weight, preferably from 0.001% by weight to 0.8% byweight, particularly preferably from 0.005% by weight to 0.3% by weight,and in particular from 0.01 to 0.2% by weight.

The agents may contain UV absorbers which attach to the treated textilesand improve the light stability of the fibers and/or the light stabilityof other formulation constituents. UV absorbers mean organic substances(light protection filters) which are able to absorb ultravioletradiation and to emit the absorbed energy again in the form of radiationof longer wavelength, for example heat.

Compounds which have these desired properties are, for example, thecompounds which are active via radiationless deactivation andderivatives of benzophenone having substituents in position(s) 2 and/or4. Furthermore, also suitable are substituted benzotriazoles, acrylateswhich are phenyl-substituted in position 3 (cinnamic acid derivatives,with or without cyano groups in position 2), salicylates, organic Nicomplexes and natural substances such as umbelliferone and theendogenous urocanic acid. Of particular importance are biphenyl andespecially stilbene derivatives, as described, for example, in EP0728749 A and commercially available as Tinosorb® FD or Tinosorb® FR exCiba. UV-B absorbers which may be mentioned are: 3-benzylidenecamphor or3-benzylidenenorcamphor and derivatives thereof, for example3-(4-methylbenzylidene)camphor, as described in EP 0693471 B1;4-aminobenzoic acid derivatives, preferably 2-ethylhexyl4-(dimethylamino)benzoate, 2-octyl 4-(dimethylamino)benzoate and amyl4-(dimethylamino)benzoate; esters of cinnamic acid, preferably2-ethylhexyl 4-methoxycinnamate, propyl 4-methoxycinnamate, isoamyl4-methoxycinnamate, 2-ethylhexyl 2-cyano-3,3-phenylcinnamate(octocrylenes); esters of salicylic acid, preferably 2-ethylhexylsalicylate, 4-isopropylbenzyl salicylate, homomenthyl salicylate;derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone,2-hydroxy-4-methoxy-4′-methylbenzophenone,2,2′-dihydroxy-4-methoxybenzo-phenone; esters of benzalmalonic acid,preferably di-2-ethylhexyl 4-methoxybenzmalonate; triazine derivativessuch as, for example,2,4,6-trianilino-(p-carbo-2′-ethyl-l′-hexyloxy)-1,3,5-triazine andoctyltriazone, as described in EP 0818450 A1, ordioctylbutamidotriazones (Uvasorb® HEB); propane-1,3-diones such as, forexample, 1-(4-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione;ketotricyclo(5.2.1.0)decane derivatives, as described in EP 0694521 B1.Further suitable are 2-phenylbenzimidazole-5-sulfonic acid and itsalkali metal, alkaline earth metal, ammonium, alkylammonium,alkanolammonium and glucammonium salts; sulfonic acid derivatives ofbenzophenones, preferably 2-hydroxy-4-methoxybenzophenone-5-sulfonicacid and its salts; sulfonic acid derivatives of 3-benzylidenecamphor,such as, for example, 4-(2-oxo-3-bornylidenemethyl)benzenesulfonic acidand 2-methyl-5-(2-oxo-3-bornylidene)sulfonic acid and salts thereof.

Suitable typical UV-A filters are, in particular, derivatives ofbenzoylmethane, such as, for example,1-(4′-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione,4-tert-butyl-4′-methoxydibenzoylmethane (Parsol 1789),1-phenyl-3-(4′-isopropylphenyl)propane-1,3-dione, and enamine compounds,as described in DE 19712033 A1 (BASF). The UV-A and UV-B filters may ofcourse also be used in mixtures. In addition to said soluble substances,insoluble light protection pigments, namely finely dispersed, preferablynanoized, metal oxides or salts, are also suitable for this purpose.Examples of suitable metal oxides are, in particular, zinc oxide andtitanium dioxide and also oxides of iron, zirconium, silicon, manganese,aluminum and cerium, and mixtures thereof. Salts which may be used aresilicates (talc), barium sulfate or zinc stearate. The oxides and saltsare already used in the form of the pigments for skin-care andskin-protective emulsions and decorative cosmetics. The particles hereshould have an average diameter of less than 100 nm, preferably between5 and 50 nm, and in particular between 15 and 30 nm. They can have aspherical shape, but it is also possible to use particles which have anellipsoidal shape or a shape deviating in some other way from thespherical form. The pigments may also be surface-treated, i.e.hydrophilicized or hydrophobicized. Typical examples are coated titaniumdioxides such as, for example, titanium dioxide T 805 (Degussa) orEusolex® T2000 (Merck); suitable hydrophobic coating agents are herepreferably silicones and, particularly preferably, trialkoxyoctylsilanesor simethicones. Preference is given to using micronized zinc oxide.Further suitable UV light protection filters can be found in the reviewby P. Finkel in SÖFW-Journal 122 (1996), p. 543.

The UV absorbers are usually used in amounts of from 0.01% by weight to5% by weight, preferably from 0.03% by weight to 1% by weight.

The ingredients usual for detergents and cleaning agents usually alsoinclude detersive and, respectively, cleaning-active enzymes. At thesame time, detergents or cleaning agents which are additionallycharacterized by further enzymes in addition to a protein of theinvention are preferred embodiments of the present invention. Examplesof these include other proteases but also oxidoreductases, cutinases,esterases and/or hemicellulases, and particularly preferably lipases,amylases, cellulases and/or β-glucanases.

Enzymes such as proteases, amylases, lipases or cellulases have beenused for decades as active components in detergents and cleaning agents.Their particular contribution to the washing and, respectively, cleaningperformance of the agent in question is, in the case of protease, theability to break down proteinaceous soilings, in the case of amylase,the breaking-down of starch-containing soilings and, in the case oflipase, fat-cleaving activity. Cellulases are preferably used indetergents, in particular due to their contribution to the secondarywashing performance of a detergent and due to their fiber action ontextiles, in addition to their soil-removing, i.e. primary washing andcleaning performance. The particular hydrolytic products are attacked,dissolved, emulsified or suspended by the other detergent or cleaningagent components or are, due to their greater solubility, washed awaywith the wash liquor, resulting in synergistic effects between theenzymes and the other components.

Proteases can exert an effect on natural fibers, in particular on woolor silk, which is comparable to the contribution by cellulase to thesecondary washing performance of an agent. Due to their action on thesurface structure of such fabrics, they can exert a smoothing influenceon the material and thereby counteract felting.

Other enzymes extend the cleaning performance of appropriate agents bytheir in each case specific enzyme performance. Examples of this includeβ-glucanases (WO 99/06515 and WO 99/06516), laccases (WO 00/39306) orpectin-dissolving enzymes (WO 00/42145) which are used, in particular,in special detergents.

Enzymes suitable for use in agents of the invention are primarily thosefrom microorganisms such as bacteria or fungi. They are obtained fromsuitable microorganisms in a manner known per se by means offermentation processes which are described, for example, in GermanLaid-Open Specifications DE 1940488, and DE 2121397, the U.S. Pat. Nos.3,623,957, 4264738, European patent application EP 006638 andinternational patent application WO 91/02792.

Particularly during storage, a protein of the invention and/or otherproteins present may be protected by stabilizers from, for example,denaturation, decay or inactivation, for example by physical influences,oxidation or proteolytic cleavage.

One group of stabilizers are reversible protease inhibitors whichdissociate off when diluting the agent in the wash liquor. Benzamidinehydrochloride and leupeptin are established for this purpose.Frequently, borax, boric acids, boronic acids or salts or esters thereofare used, including especially derivatives with aromatic groups, forexample, according to WO 95/12655, ortho-substituted, according to WO92/19707, meta-substituted and, according to U.S. Pat. No. 5,972,873,para-substituted phenylboronic acids, or salts or esters thereof. Theapplications WO 98/13460 and EP 583534 disclose peptide aldehydes, i.e.oligopeptides with reduced C terminus, that is those of 2-50 monomers,for the reversible inhibition of detergent and cleaning agent proteases.The peptidic reversible protease inhibitors include, inter alia,ovomucoid (WO 93/00418). For example, the application WO 00/01826discloses specific reversible peptide inhibitors of the proteaseSubtilisin for use in protease-containing agents, and WO 00/01831discloses corresponding fusion proteins of protease and inhibitor.

Further enzyme stabilizers are amino alcohols such as mono-, di-,triethanol- and -propanolamine and mixtures thereof, aliphaticcarboxylic acids up to C₁₂, as disclosed, for example, by theapplications EP 0378261 and WO 97/05227, such as succinic acid, otherdicarboxylic acids or salts of said acids. The application DE 19650537discloses end group-capped fatty amide alkoxylates for this purpose. Asdisclosed in WO 97/18287, particular organic acids used as builders arecapable of additionally stabilizing a contained enzyme.

Lower aliphatic alcohols, but especially polyols such as, for example,glycerol, ethylene glycol, propylene glycol or sorbitol, are otherfrequently used enzyme stabilizers. Calcium salts are also used, suchas, for example, calcium acetate or the calcium formate disclosed forthis purpose in EP 0028865, and magnesium salts, for example accordingto the European Application EP 0378262.

Polyamide oligomers (WO 99/43780) or polymeric compounds such as lignin(WO 97/00932), water-soluble vinyl copolymers (EP 828 762) or, asdisclosed in EP 702 712, cellulose ethers, acryl polymers and/orpolyamides stabilize the enzyme preparation inter alia against physicalinfluences or pH fluctuations. Polyamine N-oxide-containing polymers (EP587550 and EP 581751) simultaneously act as enzyme stabilizers and ascolor transfer inhibitors. Other polymeric stabilizers are the linearC₈-C₁₈ polyoxyalkylenes disclosed, in addition to other components, inWO 97/05227. As in the applications WO 97/43377 and WO 98/45396,alkylpolyglycosides could stabilize the enzymic components of the agentof the invention and even increase their performance. CrosslinkedN-containing compounds, as disclosed in WO 98/17764, fulfill a doublefunction as soil release agents and as enzyme stabilizers. Hydrophobic,nonionic polymer acts in a mixture together with other stabilizers,according to the application WO 97/32958, in a stabilizing manner on acellulase so that those or similar components may also be suitable forthe enzyme essential to the invention.

As disclosed inter alia in EP 780466, reducing agents and antioxidantsincrease the stability of the enzymes against oxidative decay.Sulfur-containing reducing agents are disclosed, for example, in EP0080748 and EP 0080223. Other examples are sodium sulfite (EP 533239)and reducing sugars (EP 656058).

Frequently used are also combinations of stabilizers, for example ofpolyols, boric acid and/or borax in the application WO 96/31589, thecombination of boric acid or borate, reducing salts and succinic acid orother dicarboxylic acids in the application EP 126505 or the combinationof boric acid or borate with polyols or polyamino compounds and withreducing salts, as disclosed in the application EP 080223. According toWO 98/13462, the action of peptide-aldehyde stabilizers is increased bycombination with boric acid and/or boric acid derivatives and polyolsand, according to WO 98/13459, still further increased by the additionaluse of calcium ions.

Agents containing stabilized enzyme activities are preferred embodimentsof the present invention. Particular preference is given to thosecontaining enzymes stabilized in a plurality of the manners indicated.

Since agents of the invention can be provided in any conceivable form,enzymes or proteins of the invention in any formulations appropriate foraddition to the particular agents are respective embodiments of thepresent invention. Examples thereof include liquid formulations, solidgranules or capsules.

The encapsulated form is a way of protecting the enzymes or otheringredients against other components such as, for example, bleaches, orof making possible a controlled release. Depending on their size, saidcapsules are divided into milli-, micro- and nanocapsules, microcapsulesbeing particularly preferred for enzymes. Such capsules are disclosed,for example, in the patent applications WO 97/24177 and DE 199 18 267. Apossible encapsulation method is to encapsulate the proteins, startingfrom a mixture of the protein solution with a solution or suspension ofstarch or a starch derivative, in this substance. German application DE199 56 382 entitled Verfahren zur Herstellung von mikroverkapseltenEnzymen [Method for preparing microencapsulated enzymes] describes suchan encapsulation method.

In the case of solid agents, the proteins may be used, for example, indried, granulated and/or encapsulated form. They may be addedseparately, i.e. as a separate phase, or together with other componentsin the same phase, with or without compaction. If microencapsulatedenzymes have to be processed in solid form, it is possible to remove thewater from the aqueous solutions resulting from the work-up by usingmethods known in the prior art, such as spray drying, removing bycentrifugation or resolubilizing. The particles obtained in this way areusually between 50 and 200 μgm in size.

It is possible to add to liquid, gel-like or paste-like agents of theinvention the enzymes and also the protein important to the invention,starting from protein recovery carried out according to the prior art,and preparation in a concentrated aqueous or nonaqueous solution,suspension or emulsion, but also in gel form or encapsulated or as driedpowder. Such detergents or cleaning agents of the invention are usuallyprepared by simply mixing the ingredients which may be introduced assolids or as solution into an automated mixer.

Apart from the primary washing performance, the proteases present indetergents may further fulfill the function of activating, or, after anappropriate period of action, inactivating other enzymic components byproteolytic cleavage, as disclosed, for example, in the applications WO94/29426 and EP 747 471. Comparable regulatory functions are alsopossible via the enzyme of the invention. Another embodiment of thepresent invention relates to those agents containing capsules ofprotease-sensitive material, which capsules are hydrolyzed, for example,by proteins of the invention at the intended time and release theircontents. A comparable effect may also be achieved in other multi-phaseagents.

Agents for the treatment of textile raw materials or for textile care,which are characterized in that they contain a proteolytic enzyme of theinvention, either alone or in addition to other ingredients, are aseparate subject matter of the invention, since natural fibers inparticular, such as wool or silk, for example, are distinguished by acharacteristic, microscopic surface structure. Said surface structurecan, in the long term, result in undesired effects such as, for example,felting, as discussed by way of example for wool in the article by R.Breier in Melliand Textilberichte from 4.1.2000 (p. 263). In order toavoid such effects, the natural raw materials are treated with agents ofthe invention which contribute, for example, to smoothing the flakedsurface structure based on protein structures and thereby counteractfelting. Agents of this kind for fibers or textiles containing naturalcomponents and, very particularly, containing wool or silk are aparticularly preferred embodiment.

In one embodiment of the present invention, the agent containing aprotease of the invention is designed in such a way that it can be usedregularly as a care agent, for example by adding it to the washingprocess, applying it after washing or independently of the washing. Thedesired effect is to obtain a smooth surface structure of the textileand/or to prevent and/or reduce damage to the fabric.

Methods for machine cleaning of textiles or of hard surfaces, whichmethods are characterized in that a proteolytic enzyme of the inventionbecomes active in at least one of the method steps, are a separatesubject matter of the invention.

Methods for machine cleaning of textiles are generally distinguished byseveral method steps comprising applying various cleaning-activesubstances to the material to be cleaned and, after the time of action,washing them off, or by the material to be cleaned being treated in anyother way with a cleaning agent or a solution of said agent. The sameapplies to methods for machine cleaning of any other materials astextiles which are classified under the term hard surfaces. It ispossible to add proteins of the invention to at least one of the methodsteps of such methods, which methods then become embodiments of thepresent invention.

Preference is given to methods in which an enzyme of the invention isused in an amount of from 40 μg to 4 g and, more preferably, from 50 μgto 3 g, from 100 μg to 2 g, from 200 μg to 1 g and, particularlypreferably, from 400 μg to 400 mg per application.

Since the enzyme of the invention already by nature possesses aprotein-dissolving activity and also exhibits said activity in mediawhich otherwise have no cleaning power, such as, for example, in merebuffer, an individual partial step of such a method for machine cleaningof textiles may consist of applying, if desired in addition tostabilizing compounds, salts or buffer substances, the enzyme of theinvention as single cleaning-active component. This is a particularlypreferred embodiment of the present invention.

Methods for the treatment of textile raw materials or textile care,which methods are characterized in that a proteolytic enzyme of theinvention becomes active in at least one of the method steps, are aseparate subject matter of the invention. They may be, for example,methods in which materials are prepared for use in textiles, for examplefor anti-felt finishing, or, for example, methods which add a carecomponent to the cleaning of worn textiles. Due to the above-describedaction of proteases on particular fabrics, particular embodimentscomprise textile raw materials or textiles containing naturalcomponents, in particular containing wool or silk.

The use of a proteolytic enzyme of the invention for cleaning textilesor hard surfaces is a separate subject matter of the invention, sinceenzymes of the invention may be used, in particular according to theabove-described methods, in order to remove proteinaceous soilings fromtextiles or from hard surfaces. The use outside a machine-based method,for example in manual laundry or manual removal of stains from textilesor from hard surfaces are preferred embodiments.

Preference is given to using an enzyme of the invention in an amount offrom 40 μg to 4 g and, more preferably, from 50 μg to 3 g, from 100 μgto 2 g, from 200 μg to 1 g and, particularly preferably, from 400 μg to400 mg per application.

The use of a proteolytic enzyme of the invention for activating ordeactivating ingredients of detergents or cleaning agents is a separatesubject matter of the invention, since protein components of detergentsor cleaning agents, as is known, can be inactivated by the action of aprotease. The present invention relates to specifically using thisotherwise rather undesired effect. It is likewise possible thatproteolysis only activates another component, for example if saidcomponent is a hybrid protein of the actual enzyme and the correspondinginhibitor, as disclosed, for example, in the application WO 00/01831.Another example of a regulation of this kind is one in which an activecomponent, in order to protect or control its activity, has beenencapsulated in a material susceptible to proteolytic attack. Proteinsof the invention can thus be used for inactivation reactions, activationreactions or release reactions.

The use of a proteolytic enzyme of the invention for biochemical ormolecular-biological analysis, in particular within the framework of anenzymic analytical method, is a separate subject matter of theinvention. According to the invention and according to Rbmpp, “LexikonChemie” (Version 2.0, Stuttgart/New York: Georg Thieme Verlag, 1999),enzymic analysis means any biochemical analysis which uses specificenzymes or substrates in order to determine, on the one hand, theidentity or concentration of substrates or, on the other hand, theidentity or activity of enzymes. Areas of application are any areas ofwork related to biochemistry. A preferred embodiment of this subjectmatter of the invention is the use for determining the terminal groupsin a sequence analysis.

The use of a proteolytic enzyme of the invention for the preparation,purification or synthesis of natural substances or biological valuablesubstances is a separate subject matter of the invention. Thus, it maybe necessary, for example, within the course of purifying naturalsubstances or biological valuable substances to remove from saidsubstances protein contaminations, examples of which are low molecularweight compounds, any cellular constituents or storage substances orproteins. This may be carried out both on the laboratory scale and theindustrial scale, for example after biotechnological production of avaluable substance.

A proteolytic enzyme of the invention is used for the synthesis ofproteins or other low molecular weight chemical compounds by reversingthe reaction which they catalyze by nature, for example when it isintended to link protein fragments to one another or to bind amino acidsto a compound which is not predominantly composed of protein. Possibleuses of this kind are introduced, for example, in the application EP380362.

The use of a proteolytic enzyme of the invention for the treatment ofnatural raw materials is a separate subject matter of the invention, ifit is intended to remove protein contaminations from said raw materials,which mean primarily raw materials which are obtainednon-microbiologically, for example those from agriculture.

A preferred embodiment is the use for the treatment of surfaces, andvery particularly in a method for the treatment of the economicallyimportant raw material leather. Thus, water-soluble proteins are removedfrom the skin material with the aid of proteolytic enzymes during thetanning process, in particular in the step of alkaline steep (Römpp,“Lexikon Chemie”, Version 2.0, Stuttgart/New York: Georg Thieme Verlag,1999). Proteases of the invention are suitable for this, in particularunder alkaline conditions and in the presence of denaturing agents.

The use of a proteolytic enzyme of the invention for the obtainment ortreatment of raw materials or intermediates in the manufacture oftextiles is a separate subject matter of the invention. An examplethereof is the work-up of cotton from which capsule components need tobe removed in a process referred to as sizing; another example is thetreatment of wool; the processing of raw silk is also similar. Enzymicmethods are superior to comparable chemical methods, in particular withrespect to their environmental compatibility.

In a preferred embodiment, proteins of the invention are used forremoving protective layers from textiles, in particular fromintermediate products or valuable substances, or smoothing theirsurface, before further treatment in a subsequent processing step.

In a separate subject matter of the invention, proteins of the inventionare used for the treatment of textile raw materials or for textile care,in particular for the treatment of surfaces of wool or silk or of wool-or silk-containing mixed textiles. This applies both to the preparationfor such textiles and to the care during usage, for example inconnection with the cleaning of textiles (see above).

The use of a proteolytic enzyme of the invention for the treatment ofphotographic films, in particular for removing gelatin-containing orsimilar protective layers, is a separate subject matter of theinvention, since films such as, for example, X-ray films, are coatedwith such protective layers, in particular those made of silversalt-containing gelatin emulsions, which films need to be removed fromthe support material after exposure. For this, proteases of theinvention may be used, in particular under alkaline or slightlydenaturing reaction conditions.

The use of a proteolytic enzyme of the invention for preparing food oranimal feed is a separate subject matter of the invention. Thusproteases have been used for the preparation of food from timeimmemorial. An example of this is the use of rennet for the maturingprocess of cheese or other milk products. A protein of the invention maybe added to or used to completely carry out such processes.Carbohydrate-rich food or food raw materials for non-nutritionalpurposes, such as, for example, flour or dextrin, may also be treatedwith appropriate proteases in order to remove accompanying proteins fromthem. A protease of the invention is suitable for those applications,too, in particular if they are intended to be carried out under alkalineor slightly denaturing conditions.

This applies accordingly for the preparation of animal feed. In additionto a complete removal of proteins, it may also be of interest here totreat the proteinaceous starting substances or substance mixtures withproteases only for a short time in order to render them more readilydigestible for domestic animals.

Cosmetic agents containing a proteolytic enzyme of the invention orcosmetic methods incorporating a proteolytic enzyme of the invention orthe use of a proteolytic enzyme of the invention for cosmetic purposes,in particular within the framework of corresponding methods or incorresponding agents, are a separate subject matter of the invention.

Since proteases also play a crucial part in the desquamation of humanskin (T. Egelrud et al., Acta Derm. Venerol., volume 71 (1991), pp.471-747), accordingly, proteases are also used as bioactive componentsin skincare products in order to support degradation of the desmosomestructures increasingly present in dry skin, for example according tothe applications WO 95/07688 and WO 99/18219. WO 97/07770, for example,describes the use of subtilisin proteases, in particular of the B.lentus alkaline protease variants described above, for cosmeticpurposes. Proteases of the invention, in particular those whose activityis controlled, for example after mutagenesis or due to addition ofappropriate substances interacting with them, are also suitable asactive components in skin- or hair-cleaning compositions or carecompositions. Particular preference is given to those preparations ofsaid enzymes, which, as described above, are stabilized, for example bycoupling to macromolecular supports (compare U.S. Pat. No. 5,230,891),and/or are derivatized by point mutations at highly allergenic positionsso that their compatibility with human skin is increased.

Accordingly, the use of proteolytic enzymes of this kind for cosmeticpurposes, in particular in appropriate agents such as, for example,shampoos, soaps or washing lotions or in care compositions provided, forexample, in the form of creams, is also included in this subject matterof the invention. The use in a peeling medicament is also included inthis claim.

EXAMPLES Example 1

Generation of the Protease of the Invention

All molecular-biological working steps follow standard methods asindicated, for example, in the manual by Fritsch, Sambrook and Maniatis“Molecular cloning: a laboratory manual”, Cold Spring Harbour LaboratoryPress, New York, 1989, or in international patent application WO92/21760.

Construction of the Mutagenesis Vector

The mutagenesis was carried out starting from the protease variant B.lentus alkaline protease M131. This variant is described in WO 92/21760and the strain according to this application, which produces it, hasbeen deposited with the American Type Culture Collection, Rockville,Md., USA under the name Bacillus licheniformis ATCC 68614. This straincontains the gene on plasmid pCB56M131 which replicates in Bacillus inan expression cassette comprising the promoter, the ribosomal bindingsite and the ATG start codon and the 22 amino-terminal amino acids ofthe alkaline protease from Bacillus licheniformis ATCC 53926 which arefused to the prepro-protein and the mutated sequence of Bacillus lentusDSM 5483 alkaline protease. The variant B. lentus alkaline protease M131has the following mutations, compared to the native sequence: S3T, V41,A188P, V193M, V199I.

For mutagenesis, the entire expression cassette was excised by means ofrestriction enzymes Bam HI and Sac I and cloned into the pUC18 vector(Amersham Pharmacia Biotech, Freiburg, Germany) which had likewise beencut with Bam HI and Sac I. The pUC18M131 vector thus obtained was thenused to carry out the following mutagenesis steps. FIG. 2 depicts thepUC18M131 vector. The DNA fragment containing the expression cassettefor B. lentus alkaline protease M131 is documented in SEQ ID NO. 1; SEQID NO. 2 depicts the amino acid sequence derived therefrom. The BamHI-SacI fragment depicted in SEQ ID NO. 1 extends over positions 1 to1771 in the pUC18M131 vector depicted in FIG. 2; the remaining vectorregions are identical to those of the starting plasmid pUC18.

Mutagenesis

First, the original sequence of Bacillus lentus DSM 5483 alkalineprotease at positions 188 and 193 was restored using the QuikChange®method from Stratagene (La Jolla, Calif., USA) according to themanufacturer's instructions. According to this system, a mutated plasmidwas generated in a polymerase reaction using two complementary primerscontaining the mutation in each case. After digesting the startingplasmid by means of DpnI, the reaction mixture was transformed into E.coli XL-1 blue. The clones obtained can, where appropriate, be readilyidentified by means of a restriction cleavage site introduced via themutation, with checking by DNA sequencing according to the chaintermination method with the aid of a conventional kit being possible ineach case.

The triplet coding for the amino acid in position 188, CCA (proline),was converted to GCC (alanine) by using the two primers 5′-TCA CAG TATGGC GCC GGG CTT GAC ATT-3′ and 5-AAT GTC AAG CCC GGC GCC ATA CTG TGA-3′,which contain, directly adjacent to the mutation, an Nar I restrictioncleavage site which does not alter the amino acid sequence.

The triplet coding for the amino acid at position 193, ATG (methionine),was converted to ATT (isoleucine) by using the two primers 5′-GGG CTTGAC ATT GTG GCA CCC GGG GTA AAC-3′ and 5′-GTT TAC CCC GGG TGC CAC AATGTC AAG CCC-3′ which contain, directly adjacent to the mutation, an XmaCI restriction site which does not alter the amino acid sequence.

A clone containing the doubly mutated plasmid then provided the templatefor subsequent mutation of the triplet at position 211, TTA (leucine) toGGA (glycine), for which the two complementary primers with thesequences 5′-ACG TAT GCT AGC GGA AAC GGT ACA TCG-3′ and 5′-CGA TGT ACCGTT TCC GCT AGC ATA CGT-3′ were used. Said sequences contain,immediately adjacent to the site of mutation, an Nhe I restriction sitewhich does not alter the amino acid sequence. The clones obtained whichproduce the expected fragments using Nhe I were then checked by DNAsequencing.

The DNA sequence of the BLAP-S3T, V4I, V199I, L211G mutant gene codingfor the complete protease is indicated in the sequence listing under SEQID NO. 3. The amino acid sequence indicated in the sequence listingunder SEQ ID NO. 4 can be derived therefrom. Due to the positionsdeviating from the wild-type enzyme of B. lentus DSM 5483, this B.lentus alkaline protease variant is referred to as B. lentus alkalineprotease S3T/V4I/V199I/L211G.

Expression of the Mutant and Protease Preparation

The expression cassette containing the mutated sequence was cloned backas Bam HI-Sac I fragment into the pCB56M131 vector, replacing thefragment depicted in SEQ ID NO. 1, and transformed into Bacillussubtilis DB104. The Bacillus subtilis DB 104 strain has the genotypehis, nprR2, nprE18, aprA3 (Kawamura, F. and Doi, R. H. (1984), J.Bacteriol., volume 160, pages 442-444). The DNA was transformed intoBacillus according to the variant described in WO 91/02792 of theprotoplast method originally developed by Chang and Cohen (1979; Molec.Gen. Genet., volume 168, pages 111-115).

Protease-positive clones obtained thereby were, after checking,incubated in 500 ml of MLBSP medium (10 g/l casitone; 20 g/l tryptone,10 g/l yeast extract, all from Becton Dickinson, Cockeysville; 5 g/lNaCl; 27 g/l sodium succinate; 100 mg/l MgSO₄*7 H₂O; 75 mg/l CaCl₂*2H₂O; 0.5 μM MnCl₂; 0.5 μM FeSO₄; 2% (w/v) glucose; 50 mM PIPES buffer(from a 1 M stock solution, pH 7.2); 75 mM KPO₄ (from a 1.5 M stocksolution, pH 7.0); pH=7.0, adjusted with KOH—and 10 μg/ml tetracycline)in 2 000-ml shaker flasks at 37° C. and 200 revolutions per minute for72 h. The supernatant obtained, after removing the cells bycentrifugation, was used for the experiments below, after determiningthe protease activity (according to the methods described in Tenside,volume 7 (1970), pp. 125-132).

Example 2

Textiles which had been soiled in a standardized manner and obtainedfrom the Eidgenössische Material-Prüfungsund-Versuchsanstalt, St.Gallen, Switzerland (EMPA) or the Wäschereiforschungsanstalt, Krefeld,Germany, were used for the following two examples. The followingstains/textiles were used in example 2: A (blood/milk/soot on cotton), B(blood/milk/ink on cotton), C (blood/milk/ink on a polyester-cottonblend) and D (egg/soot on cotton).

This test material was used to test the washing performances of variousdetergent formulations, using a launderometer. For this purpose, theliquor ratio was set in each case to 1:12, and washing was carried outat a temperature of 40° C. for 30 min. The dosage was 5.88 g of theparticular detergent per 1 of wash liquor. The water hardness was 160German hardness.

The control detergent used was a basic detergent formulation of thefollowing composition (all values in percent by weight): 4% linear alkylbenzenesulfonate (sodium salt), 4% C₁₂-C₁₈-fatty alcohol sulfate (sodiumsalt), 5.5% C₁₂-C₁₈-fatty alcohol with 7 EO, 1% sodium soap, 11% sodiumcarbonate, 2.5% amorphous sodium disilicate, 20% sodium perboratetetrahydrate, 5.5% TAED, 25% zeolite A, 4.5% polycarboxylate, 0.5%phosphonate, 2.5% foam inhibitor granules, 5% sodium sulfate, rest:water, optical brighteners, salts. Said formulation was admixed for thevarious series of experiments with the following proteases in such a waythat in each case a final concentration of 2.250 PE of proteolyticactivity per 1 wash liquor was obtained: B. lentus alkaline protease F49(WO 95/23221; manufacturer: Biozym, Kundl, Austria), Savinase®(Novozymes A/S, Bagsvaerd, Denmark) and the protease of the invention,B. lentus alkaline protease S3T/V4I/V199I/L211G.

After washing, the degree of whiteness of the washed textiles wasmeasured in comparison to that of barium sulfate, which had beennormalized to 100%. The measurement was carried out in a DatacolorSF500-2 spectrometer at 460 nm (UV blocking filter 3), 30 mm diaphragm,without gloss, D65 illuminant, 10°, d/8°. Table 3 below summarizes theresults obtained as percent reflectance, i.e. as percentages incomparison with barium sulfate together with the respective startingvalues. The averages of in each case 4 measurements are listed. Theyallow an immediate conclusion to be drawn about the contribution of theenzyme present on the washing performance of the agent used. TABLE 3Basic detergent with A B C D starting value 22.9 13.0 11.3 26.4 Controlwithout protease 34.1 18.5 15.1 42.4 B. lentus alkaline protease 47.537.4 49.5 72.8 S3T/V4I/V199I/L211G B. lentus alkaline protease 40.1 28.626.8 71.3 F49 Savinase ® 43.0 30.5 29.5 48.6 standard deviation 0.7 0.71.2 0.9

The data show that the protease of the invention exhibits distinctlyhigher contributions to the washing performances of the particularagents on all stains than the conventional proteases B. lentus alkalineprotease F49 and Savinase®.

Example 3

In addition to the stains/textiles indicated in example 2, the sample E(blood on cotton) was used here. The test textiles were studied in thesame way as in example 2 and with appropriate washing solutions in alaunderometer. The only difference compared to example 2 was the factthat washing was now carried out at a temperature of 60° C. Likewise,the series of experiments were evaluated as described in the previousexample; table 4 below shows the results. TABLE 4 Basic detergent with AB C D E starting value 23.1 13.0 11.0 26.6 14.9 Control without protease34.2 18.6 15.3 44.3 52.8 B. lentus alkaline 46.2 46.2 60.3 72.5 64.0protease S3T/V4I/V199I/L211G B. lentus alkaline 30.7 30.7 32.2 72.4 55.3protease F49 Savinase ® 35.9 35.9 36.4 56.5 54.0 standard deviation 0.90.9 1.8 0.8 1.3

As this result shows, the alkaline protease S3T/V4I/V199I/L211G of theinvention is also at the washing temperature of 60° C. superior or,within the margin of error, at least equal to the other proteasesestablished for detergents, B. lentus alkaline protease F49 andSavinase®.

Example 4

Vessels with hard, smooth surfaces were contacted in a standardized waywith (A) soft-boiled egg, (B) egg/milk, (C) starch mix and (D) groundmeat and washed at 45° C. using the normal program of a domesticdishwasher type Miele® G 676. 20 g of dishwashing agent were used perdishwashing run; the water hardness was 16° German hardness.

The dishwashing agent used had the following basic formulation (allvalues in each case in percent by weight): 55% sodium tripolyphosphate(calculated as anhydrous), 4% amorphous sodium disilicate (calculated asanhydrous), 22% sodium carbonate, 9% sodium perborate, 2% TAED, 2%nonionic surfactant, rest: water, dyes, perfume. This basic formulationwas admixed for the various experiments, with identical activities, withthe various proteases, B. lentus alkaline protease F49, Savinase® andthe protease variant of the invention, B. lentus-alkaline proteaseS3T/V4I/V199I/L211G, in such a way that in each case an activity of 10000 PE per dishwashing run was obtained. This corresponded in each caseto approx. 0.1 mg of protease protein per g of cleaning agentconcentrate.

After washing, the removal of stains A to C was determinedgravimetrically in percent. For this purpose, the difference between theweight of the soiled and then rinsed vessel and the starting weight ofsaid vessel was related to the weight difference of the unwashed vesselto the starting weight. This relation can be regarded as percentremoval. After washing, stain D was visually evaluated according to ascale from 0 (=unchanged, i.e. very heavily soiled) to 10 (=no soilingwhatsoever detectable). The results obtained are summarized in table 5below which lists the averages of in each case 9 measurements. Theyallow an immediate conclusion to be drawn about the contribution of theenzyme present to the washing performance of the agent used. TABLE 5 A BC D Basic detergent with % removal % removal % removal Score B. lentusalkaline 25.2 27.3 69.3 9.8 protease S3T/V4I/V199I/L211G B. lentusalkaline 26.2 22.4 65.2 7.6 protease F49 Savinase ® 12.5 12.0 63.3 8.4

These results show that the contribution of the B. lentus alkalineprotease S3T/V4I/V199I/L211G of the invention to the cleaningperformance of machine dishwashing agents is superior, but at leastequal, to that of the other proteases tested; and this already at acomparatively low activity used.

Example 5

As in the previous example, vessels were contacted with the same stainsaccording to a standard and washed in the same way with the in each casesame cleaning agent formulations. The only difference was the fact thatin each case 20 000 PE of the particular proteases were used. Thiscorresponded in each case to approx. 0.2 mg of protease in the cleaningagent concentrate. The results of the measurements, which were obtainedin the same way as in example 4, are summarized in table 6 below. TABLE6 A B D Basic detergent with % removal % removal Score B. lentusalkaline 35.4 37.6 9.4 protease S3T/V4I/V199I/L211G B. lentus alkaline33.2 32.7 9.1 protease F49 Savinase ® 12.4 14.0 8.7

With higher protease activities used, too, the higher contribution ofthe protease of the invention to the overall cleaning performance of theparticular agent compared to the proteases established for machinedishwashing agents, B. lentus alkaline protease F49 and Savinase®, isevident. Description of the figures FIG. 1: Amino acid sequencealignment of the B. lentus alkaline protease variant of the inventionwith the most important known subtilisins, in each case in the mature,i.e. processed, form, in which alignment: Inventive variant: InventiveB. lentus alkaline protease variant S3T/V4I/V199I/L211G; Subtilisin 309Bacillus lentus subtilisin according to WO 89/06279; Subtilisin PB92Bacillus nov. spec. 92 subtilisin according to EP 283075; SubtilisinCarlsberg Bacillus licheniformis subtilisin (1968), according to E. L.Smith et al., J. Biol. Chem., Volume 243, pp. 2184-2191; Subtilisin BPN'Bacillus amyloliquefaciens subtilisin according to J. A. Wells et al.(1983), Nucleic Acids Research, Volume 11, pp. 7911-7925; ConsensusPositions corresponding in the majority of the sequences indicated. FIG.2: Mutagenesis vector pUC18M131. The Bam HI-Sac I fragment depicted inSEQ ID NO. 1 extends therein over positions 1 to 1771; the remainingvector regions are identical to those of the starting plasmid pUC18(Amersham Pharmacia Biotech, Freiburg, Germany).

1-35. (Canceled)
 36. An alkaline protease of the subtilisin typecomprising isoleucine at position 199, glycine at position 211, and atleast one modification that contributes to stabilization; wherein eachposition corresponds to a position of the amino acid sequence ofBacillus lentus DSM 5483 subtilisin.
 37. An alkaline protease of thesubtilisin type comprising isoleucine at position 199, glycine atposition 211, and at least one of: threonine at position 3 or isoleucineat position 4; wherein each position corresponds to a position of theamino acid sequence of Bacillus lentus DSM 5483 subtilisin.
 38. Analkaline protease of the subtilisin type comprising isoleucine atposition 199, glycine at position 211, threonine at position 3 andisoleucine at position 4; wherein each position corresponds to aposition of the amino acid sequence of Bacillus lentus DSM 5483subtilisin.
 39. The alkaline protease of claim 36, wherein thesubtilisin is derived from a Bacillus.
 40. The alkaline protease ofclaim 39, wherein the Bacillus is Bacillus lentus.
 41. The alkalineprotease of claim 39, wherein the Bacillus is Bacillus lentus DSM 5483.42. The alkaline protease of claim 41 comprising the followingsubstitutions: S3T, V4I, V199I, and L211G.
 43. A polypeptide comprisingthe amino acid sequence of SEQ ID NO:4 or a fragment of the amino acidsequence of SEQ ID NO:4.
 44. A protein derived from the alkalineprotease of claim 36 by at least one of: fragmentation mutagenesis,deletion mutagenesis, insertion mutagenesis, substitution mutagenesis orfusion of at least one part to at least one other protein.
 45. Theprotein of claim 44 wherein the protein is additionally derivatized. 46.The protein of claim 44 wherein the protein has proteolytic activity.47. The protein of claim 46 wherein the protein has increasedproteolytic activity compared to the starting alkaline protease.
 48. Theprotein of claim 44 wherein the protein has enhanced performancecompared to the starting alkaline protease.
 49. The protein of claim 44wherein the protein is additionally stabilized.
 50. An isolated nucleicacid molecule comprising a nucleotide sequence coding for the protein ofclaim
 36. 51. An isolated nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:3 or a fragment of the nucleotidesequence of SEQ ID NO:3.
 52. A vector comprising the nucleic acidmolecule of claim
 51. 53. The vector of claim 52 wherein the vector is acloning vector.
 54. The vector of claim 52 wherein the vector is anexpression vector.
 55. A cell comprising the vector of claim
 52. 56. Ahost cell capable of expressing the alkaline protease of claim
 36. 57.The host cell of claim 56 wherein the cell is a bacterium capable ofsecreting the alkaline protease.
 58. The host cell of claim 56 whereinthe bacterium is of the genus Bacillus.
 59. The host cell of claim 56wherein the bacterium is Bacillus lentus, Bacillus licheniformis,Bacillus amyloliquefaciens, Bacillus subtilis, or Bacillus alcalophilus.60. The host cell of claim 56 wherein the cell is a eukaryotic cell. 61.The host cell of claim 56 wherein the cell is capable of modifyingpostranslationally the alkaline protease expressed.
 62. A method forpreparing an alkaline protease comprising culturing the cell of claim 56under conditions conducive to the expression of the alkaline protease.63. A composition comprising the alkaline protease of claim 36 and adetergent or cleaning agent.
 64. The composition of claim 63 wherein thealkaline protease is present in an amount of from about 2 μg to about 20mg per g of the composition.
 65. The composition of claim 63 furthercomprising one or more of: additional proteases, amylases, cellulases,hemicellulases or lipases.
 66. A composition for the treatment oftextiles or textile raw materials comprising the alkaline protease ofclaim
 36. 67. A method for cleaning textiles or surfaces comprising thestep of activating the alkaline protease of claim
 36. 68. The method ofclaim 67 wherein the alkaline protease is activated in an amount of fromabout 40 μg to about 4 g per application.
 69. The method of claim 67wherein the alkaline protease is activated in an amount of from about400 μg to about 400 mg per application.
 70. A method for the treatmentof textiles or textile raw materials comprising activating the alkalineprotease of claim
 36. 71. The method of claim 70 wherein the textile ortextile raw material being treated comprises at least one naturalcomponent.
 72. The method of claim 71 wherein the natural componentcomprises at least one of wool or silk.
 73. A method comprisingactivating or deactivating at least one detergent or cleaning agentingredient, wherein the ingredient is activated or deactivated by thealkaline protease of claim
 36. 74. A method comprising synthesizing orbiochemically analyzing a compound using the alkaline protease of claim36.
 75. A method comprising at least one of preparing, purifying orsynthesizing a biological substance using the alkaline protease of claim36.
 76. A method for the treatment of raw materials or intermediates inthe manufacture of textiles comprising the step of removing a protectivelayer on a fabric, the step comprising contacting the layer with thealkaline protease of claim
 36. 77. A method for the treatment ofphotographic films comprising the step of removing a protective layer ona film, the step comprising contacting the layer with the alkalineprotease of claim
 36. 78. A method for preparing food or animal feedcomprising treating at least one of food, animal feed, or a startingsubstance of food or animal feed with the alkaline protease of claim 36.79. A cosmetic composition comprising the alkaline protease of claim 36and a suitable carrier.